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A genomics approach to identify susceptibilities of breast cancer cells to “fever-range” hyperthermia

  • Clarissa Amaya1,
  • Vittal Kurisetty1,
  • Jessica Stiles1,
  • Alice M Nyakeriga1,
  • Arunkumar Arumugam1,
  • Rajkumar Lakshmanaswamy1,
  • Cristian E Botez2,
  • Dianne C Mitchell1 and
  • Brad A Bryan1Email author
BMC Cancer201414:81

DOI: 10.1186/1471-2407-14-81

Received: 31 July 2013

Accepted: 22 January 2014

Published: 11 February 2014

Abstract

Background

Preclinical and clinical studies have shown for decades that tumor cells demonstrate significantly enhanced sensitivity to “fever range” hyperthermia (increasing the intratumoral temperature to 42-45°C) than normal cells, although it is unknown why cancer cells exhibit this distinctive susceptibility.

Methods

To address this issue, mammary epithelial cells and three malignant breast cancer lines were subjected to hyperthermic shock and microarray, bioinformatics, and network analysis of the global transcription changes was subsequently performed.

Results

Bioinformatics analysis differentiated the gene expression patterns that distinguish the heat shock response of normal cells from malignant breast cancer cells, revealing that the gene expression profiles of mammary epithelial cells are completely distinct from malignant breast cancer lines following this treatment. Using gene network analysis, we identified altered expression of transcripts involved in mitotic regulators, histones, and non-protein coding RNAs as the significant processes that differed between the hyperthermic response of mammary epithelial cells and breast cancer cells. We confirmed our data via qPCR and flow cytometric analysis to demonstrate that hyperthermia specifically disrupts the expression of key mitotic regulators and G2/M phase progression in the breast cancer cells.

Conclusion

These data have identified molecular mechanisms by which breast cancer lines may exhibit enhanced susceptibility to hyperthermic shock.

Keywords

Breast cancer Hyperthermia Heat shock Microarray Genomics Gene expression

Background

Although the effectiveness of standard therapies such as surgery, chemotherapy, and irradiation has steadily improved over the years, cancer remains one of the most challenging problems of modern medicine. Among the major issues that complicate cancer treatment is the fact that cancerous cells are very difficult to therapeutically target with any specificity as they are in many respects similar to normal cells and have an astonishing ability of “hiding” their peculiarities. It has been known for over three decades that tumor cells demonstrate significantly more sensitivity to mild hyperthermia in “fever-range” temperatures (41-45°C) than normal cells [1, 2]. Mild hyperthermia has been shown in a wealth of preclinical oncology studies to act as a dose modifying agent that increases the therapeutic ratio of conventional therapy, thus enhancing the effectiveness of a given dose without additional toxicity [3]. Furthermore, numerous clinical trials have combined hyperthermia with radiation therapy and/or chemotherapy for many types of carcinomas (including breast cancers) and sarcomas, and most studies have shown a significant reduction in tumor volume when hyperthermia is combined with standard treatments [47]. Various hyperthermia techniques have been developed to treat breast cancer, including focused ultrasound [8], focused microwaves [9, 10], and radiofrequency electric fields [11]. Despite these techniques, various factors including tumor size and depth greatly affect the homogenous distribution of heat specific to and throughout the entire tumor mass. With the recent and rapid progression of nanobiotechnology applications in medicine, the development of magnetic nanoparticles which can induce tumor hyperthermia through hysteresis loss in an alternating magnetic field has renewed great interest in reexamining this adjuvant therapy in tumor treatments [12]. Further development of this technology may have the potential to overcome the previous limitations associated with older modalities of inducing hyperthermia and lead to reduced morbidity and mortality for patients.

Hyperthermia over a short period (generally 30 minutes to 1 hour) has been shown to induce irreversible cell damage and subsequent death in tumor cells, yet normal cells are remarkably spared [1, 2]. These effects are often very rapid, with tumor apoptosis and necrosis occurring within a short time (3–6 hrs) post heating [13]. Several mechanisms have been proposed as to how hyperthermia kills tumor cells including disruption of plasma membrane protein and cytoskeletal distribution, altering mitochondrial membrane potential and cellular redox status, disrupting cell cycle progression, inducing tumor hypoxia, and affecting DNA damage repair mechanisms in the nucleus [1416], yet despite several decades of research the definitive identification of mechanisms leading to the favorable clinical results of hyperthermia have not been established. It has been further hypothesized that the strong anti-tumor effect of hyperthermia may be due to the low blood flow rate (and thus reduced dispersant cooling following heating) found in the center of tumors due to a disorganized and often dysfunctional vascular system. Additionally, several reports indicate that hyperthermia induces a strong immunological response via activation of immune cells and sensitization of tumor cells to immune effector cells [1719]. Several studies have elucidated the heat shock induced changes in global gene expression of tumor cell lines such as squamous cell carcinoma, lymphoma, and glioma and have commonly identified gene networks involved in apoptosis, cell cycle, and cell structure/maintanence [2022]. However, none of these studies compared the gene expression profiles to that of hyperthermia treated normal cells, thus it remains unknown how the hyperthermic response of cancer cells differs from that of normal cells. Identification of the unique hyperthermia-induced gene expression changes between normal and cancer cells may not only shed light on the selective disadvantage of solid tumors in response to mild increases in temperature, but could also identify signaling targets and biological processes which potentially could be exploited to sensitize tumors to chemotherapy and radiation.

To address this issue, we analyzed the hyperthermia-induced global gene expression profiles of a panel of breast cancer and mammary epithelial cell lines and used bioinformatics analysis to identify the unique gene networks distinct between the normal and cancer lines following this treatment. Furthermore, we confirmed our identified gene expression changes using qPCR and utilized flow cytometry to verify that these transcriptional alterations indeed reflect breast cancer specific responses to hyperthermia.

Methods

Cell culture and hyperthermia treatment

MCF10A (ATCC #CRL10318) mammary epithelial cells and MCF7 luminal breast cancer cells (ATCC #HTB-22), MDA-MB-231 Basal B breast cancer cells (ATCC # HTB-26), and MDA-MB-468 Basal A breast cancer cells (ATCC #HTB-132) were purchased from ATCC and grown in standard culture conditions as previously reported [2325]. For heat shock, cells were split into two groups: 37°C control (C and C’ for mammary epithelial and breast cancer cells, respectively) and 45°C hyperthermic treatment (H and H’ for mammary epithelial and breast cancer cells, respectively). The 37°C control was grown under standard culture conditions. For the hyperthermia treatment, 45°C prewarmed conditioned media was immediately added to each treatment group and continuously maintained at this temperature for 30 minutes. After this time, the 45°C media was completely removed and replaced with 37°C conditioned media. The cells were then grown under standard culture conditions and harvested at the time point indicated for each experiment.

Microarray analysis

Total RNA was collected from each cell line (triplicate biological replicates) 4 hours after completion of the hyperthermia treatment. RNA was amplified and biotin-labeled using Illumina TotalPrep RNA Amplification Kit (Ambion). 750 ng of biotinylated aRNA was then briefly heat-denatured and loaded onto expression arrays to hybridize overnight (triplicate technical replicates). Following hybridization, arrays were labeled with Cy3-streptavidin and imaged on the Illumina ISCAN. Intensity values were transferred to GeneSpring GX microarray analysis software (Agilent) and data was filtered based on quality of each call. Statistical relevance was determined using ANOVA with a Benjamini Hochberg FDR multiple testing correction (p-value < 0.05). Data were then limited by fold change analysis to statistically relevant data points demonstrating a 2-fold or more change in expression. The microarray data from this experiment is publically available on the Gene Expression Omnibus (GEO Accession #GSE48398). All heatmaps shown represent the combined average of all biological and technical replicates.

Bioinformatics analysis of microarray data

Pathway analysis to identify gene networks and biological processes affected by the gene expression changes was performed using Metacore software (Thomson Reuters). Protein-protein interaction networks were determined using String 9.05 (http://string-db.org).

Quantitative real time PCR analysis

RNA was isolated from cells 4 hours after the hyperthermia treatment using the Ambion Purelink Minikit according to the manufacturer’s directions. The RNA collected was from an independent biological experiment separate from the RNA collected for the microarray to minimize the discovery of false positives. qRT-PCR was performed on an ABI7900HT RT-PCR system using TaqMan Assays with predesigned primer sets for the genes of interest (Invitrogen). All RT-PCR experiments were performed in at least triplicate.

Flow cytometry

Cells were harvested 24 hours post treatment via trypsinization and stained with propidium iodide as previous reported [26]. Cell cycle profiles were independently obtained using either a BD LSRII flow cytometer or an Accuri C6 flow cytometer. Flow cytometry data was analyzed using FlowJo software (Tree Star) or CFlow Plus software (Accuri).

Results

Determination of the global transcriptional response of mammary epithelial and breast cancer cells to fever range hyperthermia

It remains to be determined how mild hyperthermia preferentially selects against breast cancer cells, yet largely spares normal tissue from collateral damage. To address this question, we first sought to elucidate how hyperthermia induces alterations in gene expression patterns in mammary epithelial and breast cancer cells. Mammary epithelial cells (MCF10A) and three malignant breast cancer lines from each of the known subtypes (MCF7 [luminal], MDA231 [Basal B], and MDA468 [Basal A]) were subjected to 30 minutes of fever range hyperthermic shock (or maintained at 37°C as a control) as described in the Materials and Methods section. To streamline identification of these treatment groups, cells grown at 37°C will be referred to as C and C’ (for mammary epithelial and breast cancer cells, respectively), while cells grown at 45°C will be referred to as H and H’ (for mammary epithelial and breast cancer cells, respectively). Total RNA was isolated 4 hours following hyperthermic treatment. We then performed microarray analysis of the global transcription changes using Illumina high density BeadArrays which measure the expression levels of more than 47,000 transcripts and known splice variants across the human transcriptome. Data was filtered based on quality of each call and statistical relevance was determined using ANOVA with a Benjamini Hochberg FDR multiple testing correction (p-value < 0.05). Data were then limited by fold change analysis to statistically relevant data points demonstrating a 2-fold or more change in expression. When comparing the expression changes based on the C vs H and C’ vs H’ analysis, we discovered that hyperthermia induced very dramatic changes in gene expression in all cell lines tested as reflected by 7252 two-fold or greater statistically significant gene expression changes (p < 0.05) occurring in at least one of the four cell lines (Figure 1A). Specifically, hyperthermia significantly altered the expression of 2670 genes in the MCF10A line (1810 genes upregulated and 860 genes downregulated), 442 genes in MCF7 (72 genes upregulated and 370 genes downregulated), 615 genes in MDA231 (244 genes upregulated and 371 downregulated), and 4458 genes in MDA468 (1744 genes upregulated and 2714 genes downregulated). A list of the top and bottom most regulated genes for each cell line can be found in Table 1. The complete gene expression dataset has been freely and publically deposited in Gene Expression Omnibus for ease of access and meta-analysis (GEO Accession #48398). These data suggest that mild hyperthermia induces large-scale alterations in gene expression profiles across normal and breast cancer cell lines.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-81/MediaObjects/12885_2013_Article_4319_Fig1_HTML.jpg
Figure 1

Fever range hyperthermic shock induces large-scale changes in gene expression in breast cancer and mammary epithelial cells. (A) Heatmap depicting the 7252 two-fold or greater changes in gene expression (p < 0.05) occurring in the C vs H and C’ vs H’ comparisons. Hierarchical clustering based on cell lines shows the degree of similarity with respect to gene expression clustering for each indicated cell line (red?=?overexpressed, green?=?underexpressed). (B) Venn diagram illustrating common and unique 2-fold or greater gene expression changes (p < 0.05) between each of the cell lines in the C vs H and C’ vs H’ comparison. (C) Profile plot of the normalized intensity values for each two-fold or greater gene expression change (p < 0.05) showing relative expression for each cell line in the in the C vs H and C’ vs H’ comparison.

Table 1

List of the top and bottom most regulated genes for each cell line in the C vs H and C’ vs H’ comparisons

Gene symbol

Gene name

Accession number

MCF-10A

MCF-7

MDA-231

MDA-468

MCF10A

      

XAGE1A

X antigen family, member 1A, TV3

NM_001097593.1

64.0

1.2

1.0

122.0

XAGE1B

X antigen family, member 1B, TV1

NM_001097595.1

60.3

1.2

1.1

131.6

SRGN

Serglycin, TV1

NM_002727.2

58.1

1.1

-1.5

65.8

SRGN

Serglycin, TV1

NM_002727.2

56.5

1.2

-1.5

67.7

LOC652683

Similar to sperm protein associated with the nucleus, X chromosome, family member B1

XM_942283.2

50.0

1.2

-1.0

71.5

SPANXA2

SPANX family, member A2

NM_145662.2

49.3

1.2

-1.1

67.7

SPANXB1

SPANX family, member B1

NM_032461.2

48.7

1.2

-1.0

70.0

TOP2A

Topoisomerase (DNA) II alpha 170 kDa

NM_001067.2

39.0

-2.0

-3.7

1.1

LOC653219

Similar to G antigen, family D, 2 isoform 1a, TV1

XM_927237.1

37.0

1.2

1.1

71.8

LOC100133171

Hypothetical protein LOC100133171

XM_001713741.1

34.8

1.3

-1.1

52.7

RN5S9

RNA, 5S ribosomal 9

NR_023371.1

-48.2

11.5

59.7

11.5

RN7SK

RNA, 7SK small nuclear

NR_001445.1

-55.1

13.1

45.4

38.6

SERPINB5

Serpin peptidase inhibitor, clade B (ovalbumin), member 5

NM_002639.3

-65.8

1.2

7.6

-3.8

KIAA1666

KIAA1666 protein

XM_942124.2

-72.9

17.9

33.4

35.3

AKR1C2

Aldo-keto reductase family 1, member C2, TV1

NM_001354.4

-82.3

1.4

24.4

-9.6

LOC650517

Hypothetical LOC650517

XR_019109.1

-90.0

1.2

1.7

-2.0

RN7SK

RNA, 7SK small nuclear

NR_001445.1

-109.9

20.2

62.1

45.2

KRT17P3

Predicted misc_RNA KRT17P3

XR_015626.2

-133.2

1.5

2.6

-4.5

LOC651397

Predicted misc_RNA LOC651397

XR_037048.1

-141.4

1.2

5.1

-4.1

KRT6A

Keratin 6A

NM_005554.3

-242.8

1.2

116.7

-11.7

MCF7

      

RNY5

RNA, Ro-associated Y5

NR_001571.2

-17.4

22.6

4.6

4.3

RN7SK

RNA, 7SK small nuclear

NR_001445.1

-109.9

20.2

62.1

45.2

KIAA1666

KIAA1666 protein

XM_942124.2

-72.9

17.9

33.4

35.3

MIR1974

MicroRNA 1974

NR_031738.1

-9.4

14.2

20.8

133.3

LOC100008589

28S ribosomal RNA

NR_003287.1

-26.8

13.8

26.4

113.8

RN7SK

RNA, 7SK small nuclear

NR_001445.1

-55.1

13.1

45.4

38.6

LOC100132394

Hypothetical protein LOC100132394

XM_001713809.1

-19.4

12.3

22.2

105.0

RNU4-1

RNA, U4 small nuclear 1

NR_003925.1

-7.7

11.5

11.4

13.2

RN5S9

RNA, 5S ribosomal 9

NR_023371.1

-48.2

11.5

59.7

11.5

SNORD3C

Small nucleolar RNA, C/D box 3C

NR_006881.1

-34.9

11.1

30.5

11.8

CCDC117

Coiled-coil domain containing 117

NM_173510.1

3.2

-3.3

-1.7

-1.8

LOC730432

Hypothetical protein LOC730432

XM_001125680.1

3.7

-3.3

-1.3

-4.7

LOC644799

Hypothetical protein LOC644799, TV1

XM_934554.1

2.5

-3.3

-2.1

1.1

RPS6KB1

Ribosomal protein S6 kinase, 70 kDa, polypeptide 1

NM_003161.2

2.9

-3.4

-1.8

-3.7

NRIP1

Nuclear receptor interacting protein 1

NM_003489.2

1.1

-3.4

-1.8

-4.2

AP4E1

Adaptor-related protein complex 4, epsilon 1 subunit

NM_007347.3

2.3

-3.4

-2.1

-2.2

DCP2

DCP2 decapping enzyme homolog (S. cerevisiae), TV1

NM_152624.4

3.1

-3.4

-2.4

-6.8

TROVE2

TROVE domain family, member 2, TV1

NM_001042369.1

2.0

-3.8

-1.2

-8.2

PURB

Purine-rich element binding protein B

NM_033224.3

3.2

-3.8

-2.6

-7.1

ZNF217

Zinc finger protein 217

NM_006526.2

3.2

-3.9

-1.7

-2.5

MDA231

      

KRT6A

Keratin 6A

NM_005554.3

-242.8

1.2

116.7

-11.7

RN7SK

RNA, 7SK small nuclear

NR_001445.1

-109.9

20.2

62.1

45.2

RN5S9

RNA, 5S ribosomal 9

NR_023371.1

-48.2

11.5

59.7

11.5

RN7SK

RNA, 7SK small nuclear

NR_001445.1

-55.1

13.1

45.4

38.6

SNORD3D

Small nucleolar RNA, C/D box 3D

NR_006882.1

-47.2

11.1

36.8

9.4

SNORD3A

Small nucleolar RNA, C/D box 3A

NR_006880.1

-46.0

11.1

36.5

9.0

KIAA1666

KIAA1666 protein

XM_942124.2

-72.9

17.9

33.4

35.3

SNORD3C

Small nucleolar RNA, C/D box 3C

NR_006881.1

-34.9

11.1

30.5

11.8

LOC100008589

28S ribosomal RNA

NR_003287.1

-26.8

13.8

26.4

113.8

LOC100132564

Hypothetical protein LOC100132564

XM_001713808.1

-21.6

8.4

25.3

13.3

PAK2

p21 protein (Cdc42/Rac)-activated kinase 2

NM_002577.3

5.9

-2.9

-3.1

-1.3

CEP55

Centrosomal protein 55 kDa, TV1

NM_018131.3

19.2

-1.7

-3.2

-1.2

RHOBTB3

Rho-related BTB domain containing 3

NM_014899.3

14.0

-1.1

-3.2

2.5

ZAK

Sterile alpha motif and leucine zipper containing kinase AZK, V2

NM_133646.2

2.8

-2.6

-3.2

-4.0

KATNAL1

Katanin p60 subunit A-like 1, TV2

NM_001014380.1

3.6

-1.4

-3.2

-1.3

PBK

PDZ binding kinase

NM_018492.2

10.5

-1.9

-3.3

-1.0

PAFAH1B1

Platelet-activating factor acetylhydrolase, isoform Ib, alpha subunit

NM_000430.2

4.5

-2.6

-3.5

1.2

TOP2A

Topoisomerase (DNA) II alpha 170 kDa

NM_001067.2

39.0

-2.0

-3.7

1.1

FAM83D

Family with sequence similarity 83, member D

NM_030919.2

15.4

-1.8

-4.0

-1.7

BCAT1

Branched chain aminotransferase 1, cytosolic

NM_005504.4

8.9

1.1

-4.9

2.9

MDA468

      

MIR1974

microRNA 1974

NR_031738.1

-9.4

14.2

20.8

133.3

XAGE1B

X antigen family, member 1B, TV1

NM_001097595.1

60.3

1.2

1.1

131.6

XAGE1A

X antigen family, member 1A, TV3

NM_001097593.1

64.0

1.2

1.0

122.0

LOC100008589

28S ribosomal RNA

NR_003287.1

-26.8

13.8

26.4

113.8

LOC100132394

Hypothetical protein LOC100132394

XM_001713809.1

-19.4

12.3

22.2

105.0

CST3

Cystatin C

NM_000099.2

-1.8

1.2

1.7

103.9

ACTG1

Actin, gamma 1

NM_001614.2

1.1

1.5

1.5

75.2

LOC653219

Similar to G antigen, family D, 2 isoform 1a,

XM_927237.1

37.0

1.2

1.1

71.8

LOC652683

Similar to sperm protein associated with the nucleus, X chromosome, family member B1

XM_942283.2

50.0

1.2

-1.0

71.5

SPANXB1

SPANX family, member B1

NM_032461.2

48.7

1.2

-1.0

70.0

CSAG1

Chondrosarcoma associated gene 1, TVb

NM_153479.1

1.7

1.2

-1.0

-27.4

SERPINA3

Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3

NM_001085.4

-8.5

1.1

2.0

-30.5

ALDH1A3

Aldehyde dehydrogenase 1 family, member A3

NM_000693.1

-1.5

-1.0

2.0

-34.0

EPCAM

Epithelial cell adhesion molecule

NM_002354.2

1.6

-1.2

-1.2

-36.1

LAD1

Ladinin 1

NM_005558.3

-47.9

1.2

13.5

-37.5

OLFML3

Olfactomedin-like 3

NM_020190.2

-1.2

1.2

1.2

-39.0

TACSTD1

Tumor-associated calcium signal transducer 1

NM_002354.1

2.1

-1.1

-1.4

-47.5

RARRES1

Retinoic acid receptor responder (tazarotene induced) 1, TV1

NM_206963.1

-1.3

1.3

-1.1

-49.9

MGP

Matrix Gla protein

NM_000900.2

-18.6

1.0

2.4

-51.1

KLK5

Kallikrein-related peptidase 5, TV1

NM_012427.4

-1.3

1.1

1.1

-56.3

Hierarchical clustering of the gene expression changes based on each cell line indicates that the breast cancer lines responded to hyperthermia more similarly to each other than to the mammary epithelial line (Figure 1A). Using a Venn diagram that strictly eliminated any genes with less than a 2-fold expression change (p < 0.05), we compared the gene expression profiles that were shared and unique between each cell line in response to hyperthermia, revealing that while many gene expression changes were common between one or more of the breast cancer lines, not a single 2-fold or greater gene expression change was shared between the mammary epithelial line and all three breast cancer lines (Figure 1B). This data strongly suggested that the hyperthermic response of breast cancer cells is truly distinct from that of mammary epithelial cells. As an independent assessment, we generated profile plots depicting the changes in normalized intensity values between the four cell lines, revealing that many of the statistically significant gene expression alterations we identified were largely shared between the three breast cancer lines and distinctly unique from that of the MCF10A line (Figure 1C). Using Metacore network analysis of the microarray data, we identified key signaling pathways that were unique to the mammary epithelial line and the three breast cancer lines. The hyperthermic response of MFC10A was strongly indicative of statistically significant gene expression alterations in a large number of genes involved in cell cycle regulation, apoptosis, heat shock response, and DNA damage response, (Figure 2A-D, Table 2) and changes in the expression of genes involved in these biological pathways were not observed in the three breast cancer lines. Network analysis indicated that signaling pathways with the highest statistical significance amongst the three breast cancer lines responding to hyperthermia (but not in the MCF10A line) included genes involved in Ras and Rab5A G-protein regulation (GAPVD1, RASA1, RABEP1, CALM1, GMFB, PTPN11) (Figure 2E, Table 2) and survival/apoptosis (MAP2K4, BIRC2) (Figure 2B, Table 2).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-81/MediaObjects/12885_2013_Article_4319_Fig2_HTML.jpg
Figure 2

Mammary epithelial cells respond to fever range hyperthermia through transcriptional alterations in gene networks unique from that of breast cancer cells. Hierarchical clustered heatmaps depicting the transcriptional expression changes for genes involved in cell cycle (A), apoptosis (B), heatshock (C), DNA damage (D), and Ran/Rab (E) regulation in the C vs H and C’ vs H’ comparison (red?=?overexpressed, green?=?underexpressed).

Table 2

List of genes involved in the gene networks that are differentially regulated between mammary epithelial cells in the ( C vs H and C’ vs H’ ) comparison

Gene symbol

Gene name

Accession number

MCF-10A

MCF-7

MDA-231

MDA-468

DNA Damage

      

USP1

Ubiquitin specific peptidase 1, TV3

NM_001017416.1

4.8

-1.8

-2.2

-5.3

CDC25A

Cell division cycle 25A, TV1

NM_001789.2

4.6

-1.3

1.2

2.0

BARD1

BRCA1 associated RING domain 1

NM_000465.1

3.5

-1.7

-2.8

-1.7

MDC1

Mediator of DNA-damage checkpoint 1

NM_014641.1

3.0

-1.4

1.1

-1.2

NBN

Nibrin

NM_002485.4

2.5

-2.4

-2.2

-1.1

SLC35B2

Solute carrier family 35, member B2

NM_178148.1

2.3

-1.0

1.8

1.8

CHEK1

Checkpoint kinase 1, TV3

NM_001274.3

2.3

-1.1

-1.4

-2.6

CCNE1

Cyclin E1, TV2

NM_057182.1

2.1

-1.2

-1.1

-1.0

CCNA1

Cyclin A1, TV1

NM_003914.2

-2.2

1.2

1.0

-1.3

GADD45B

Growth arrest and DNA-damage-inducible, beta

NM_015675.2

-3.8

1.1

1.8

-1.1

CDKN1A

Cyclin-dependent kinase inhibitor 1A (p21, Cip1), TV1

NM_000389.2

-4.7

1.1

2.5

1.1

Cell Cycle

      

AURKA

Aurora kinase A, TV5

NM_198436.1

22.0

-1.3

-2.1

1.2

CDC20

Cell division cycle 20

NM_001255.2

18.0

1.1

-1.1

-1.2

CCNB2

Cyclin B2

NM_004701.2

17.5

-1.3

-2.3

-1.1

NCAPG

Non-SMC condensin I complex, subunit G, TV1

NM_022346.3

14.9

-1.4

-2.6

-1.1

BUB1

Budding uninhibited by benzimidazoles 1 homolog (yeast)

NM_004336.2

10.8

-1.5

-2.3

1.2

MAD2L1

MAD2 mitotic arrest deficient-like 1 (yeast)

NM_002358.2

9.8

-1.5

-1.6

2.1

CDC45L

Cell division cycle 45, TV2

NM_003504.3

8.0

1.1

1.4

1.1

CENPF

Centromere protein F, 350/400 kDa

NM_016343.3

7.8

-1.3

-3.0

1.1

PTTG1

Pituitary tumor-transforming 1

NM_004219.2

7.2

1.0

-1.0

1.4

MCM3

Minichromosome maintenance complex component 3, TV1

NM_002388.3

7.2

-1.5

-1.1

-1.4

CCNB1

Cyclin B1

NM_031966.2

6.9

-1.2

-2.5

1.1

CENPE

Centromere protein E, 312 kDa

NM_001813.2

6.6

-1.4

-2.5

-1.5

KIF11

Kinesin family member 11

NM_004523.2

6.5

-1.4

-2.7

-1.0

NDC80

NDC80 kinetochore complex component

NM_006101.1

6.5

-1.1

-1.8

1.4

XPO1

Exportin 1 (CRM1 homolog, yeast)

NM_003400.3

6.1

-2.7

-2.4

-3.0

RFC5

Replication factor C (activator 1) 5, 36.5 kDa, TV1

NM_007370.3

5.8

-1.2

-1.1

1.7

POLA1

Polymerase (DNA directed), alpha 1, catalytic subunit

NM_016937.2

5.7

-1.5

-1.8

-1.2

RFC4

Replication factor C (activator 1) 4, 37 kDa, TV2

NM_181573.1

5.6

-1.2

-1.4

-1.4

CDC2

Cyclin-dependent kinase 1, TV1

NM_001786.2

5.2

-1.2

-2.3

1.7

CENPA

Centromere protein A, TV2

NM_001042426.1

4.9

-1.3

-1.7

1.3

CKS1B

CDC28 protein kinase regulatory subunit 1B, TV1

NM_001826.1

4.8

-1.2

-1.3

1.6

DSN1

MIND kinetochore complex component, TV3

NM_024918.2

4.7

-1.3

-1.8

-1.1

CDC25A

Cell division cycle 25A, TV1

NM_001789.2

4.6

-1.3

1.2

2.0

NUF2

NDC80 kinetochore complex component, TV2

NM_031423.3

4.3

1.0

-2.0

1.7

PPP2CA

Protein phosphatase 2, catalytic subunit, alpha isozyme

NM_002715.2

4.3

-1.8

-2.0

-2.6

PRIM1

Primase, DNA, polypeptide 1 (49 kDa)

NM_000946.2

4.2

1.0

-1.0

1.8

YWHAH

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide

NM_003405.3

4.0

-1.9

-1.6

-3.9

TUBB

Tubulin, beta class I

NM_178014.2

3.9

-1.1

1.3

1.9

STAG2

Stromal antigen 2, TV2

NM_001042750.1

3.9

-2.8

-2.4

-3.4

CSE1L

CSE1 chromosome segregation 1-like (yeast), TV2

NM_177436.1

3.8

-1.4

-1.6

1.3

MCM6

Minichromosome maintenance complex component 6

NM_005915.4

3.8

-1.3

-1.4

-3.4

RPA1

Replication protein A1, 70 kDa

NM_002945.2

3.7

-1.3

-1.2

1.1

TOP2B

Topoisomerase (DNA) II beta 180 kDa

NM_001068.2

3.7

-2.5

-2.6

-2.0

RALA

V-ral simian leukemia viral oncogene homolog A (ras related)

NM_005402.2

3.6

-1.7

-1.6

-2.3

SPC25

SPC25, NDC80 kinetochore complex component

NM_020675.3

3.6

-1.1

-1.4

1.4

MAPK13

Mitogen-activated protein kinase 13, TV1

NM_002754.3

3.6

1.1

1.3

-1.2

RPA3

Replication protein A3, 14 kDa

NM_002947.3

3.5

-1.2

-1.1

-1.3

E2F3

E2F transcription factor 3, TV1

NM_001949.2

3.5

-2.0

-2.2

-4.5

MCM10

Minichromosome maintenance complex component 10, TV2

NM_018518.3

3.4

-1.2

-1.2

2.3

NEK2

NIMA-related kinase 2, TV1

NM_002497.2

3.4

-1.1

-2.1

1.1

MAP2K4

Mitogen-activated protein kinase kinase 4

NM_003010.2

3.3

-2.3

-2.5

-3.1

DYNC1H1

Dynein, cytoplasmic 1, heavy chain 1

NM_001376.2

3.3

-2.1

-1.1

1.2

KIF22

Kinesin family member 22, TV1

NM_007317.1

3.2

-1.1

-1.1

1.1

RPA2

Replication protein A2, 32 kDa

NM_002946.3

3.2

-1.0

1.0

1.4

CDC7

Cell division cycle 7, TV1

NM_003503.2

3.2

-1.3

-1.8

-3.1

RBL1

Retinoblastoma-like 1 (p107), TV1

NM_002895.2

3.2

-1.1

-2.0

1.4

MCM5

Minichromosome maintenance complex component 5

NM_006739.3

3.1

1.1

1.8

1.8

TGFB2

Transforming growth factor, beta 2

NM_003238.1

3.0

1.2

-1.4

1.7

E2F2

E2F transcription factor 2

NM_004091.2

3.0

-1.7

1.2

-3.7

CDCA1

Cell division cycle associated 1, TV1

NM_145697.1

3.0

1.1

-2.3

1.9

ITGB1

Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12), TV1

NM_002211.2

3.0

-1.7

-1.2

1.4

MCM2

Minichromosome maintenance complex component 2, TV1

NM_004526.2

3.0

1.0

1.7

1.8

RFC3

Replication factor C (activator 1) 3, 38 kDa, TV1

NM_002915.3

2.9

-1.7

-1.8

-2.2

NEDD8

Neural precursor cell expressed, developmentally down-regulated 8

NM_006156.2

2.9

-1.1

-1.0

1.4

BIRC5

Baculoviral IAP repeat containing 5, TV3

NM_001012271.1

2.9

-1.1

-1.4

1.2

RPS6KB1

Ribosomal protein S6 kinase, 70 kDa, polypeptide 1, TV1

NM_003161.2

2.9

-3.4

-1.8

-1.2

KPNA4

Karyopherin alpha 4 (importin alpha 3)

NM_002268.3

2.8

-1.3

-2.1

-1.1

PPP1CB

Protein phosphatase 1, catalytic subunit, beta isozyme, TV3

NM_206876.1

2.7

-2.6

-2.9

-1.1

PLK1

Polo-like kinase 1

NM_005030.3

2.7

1.1

-1.1

1.2

CDK6

Cyclin-dependent kinase 6, TV1

NM_001259.5

2.7

-1.5

-2.9

2.1

NEK6

NIMA-related kinase 6, TV2

NM_014397.3

2.7

1.1

-1.2

1.1

KPNB1

Karyopherin (importin) beta 1, TV1

NM_002265.4

2.7

-1.2

-1.2

1.1

RAD21

RAD21 homolog (S. pombe)

NM_006265.1

2.7

-1.7

-1.7

1.4

DSCC1

Defective in sister chromatid cohesion 1

NM_024094.1

2.7

-1.6

-1.6

-1.4

TNPO1

Transportin 1, TV2

NM_153188.2

2.7

-1.6

-2.4

1.1

YWHAB

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide, TV1

NM_003404.3

2.6

1.1

-1.1

-1.5

TUBG1

Tubulin, gamma 1

NM_001070.3

2.6

1.0

-1.0

-1.0

FBXW11

F-box and WD repeat domain containing 11, TV1

NM_033645.2

2.6

-2.3

-2.3

1.4

BUB3

Budding uninhibited by benzimidazoles 3 homolog (yeast), TV2

NM_001007793.1

2.6

1.1

-1.1

-3.0

TUBA1B

Tubulin, alpha 1b

NM_006082.2

2.6

1.1

1.2

1.7

DYNC1LI2

Dynein, cytoplasmic 1, light intermediate chain 2

NM_006141.2

2.6

-2.5

-1.5

-1.2

TUBB2A

Tubulin, beta 2A class IIa

NM_001069.2

2.5

1.3

1.5

-1.4

CHUK

Conserved helix-loop-helix ubiquitous kinase

NM_001278.3

2.5

-1.9

-2.1

1.7

IPO5

Importin 5

NM_002271.4

2.5

-1.2

-2.0

1.3

RAN

RAN, member RAS oncogene family

NM_006325.2

2.5

-1.4

-1.5

1.6

RASSF1

Ras association (RalGDS/AF-6) domain family member 1, TVA

NM_007182.4

2.5

1.2

1.4

-1.1

INCENP

Inner centromere protein antigens 135/155 kDa, TV1

NM_001040694.1

2.5

-1.2

-1.2

2.0

DYNLT3

Dynein, light chain, Tctex-type 3

NM_006520.1

2.5

-1.3

-1.8

1.7

NCAPD3

Non-SMC condensin II complex, subunit D3

NM_015261.2

2.5

-1.1

1.2

-2.6

YWHAQ

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide

NM_006826.2

2.5

-1.2

-1.4

1.8

TFDP1

Transcription factor Dp-1, TV1

NM_007111.3

2.4

-1.4

-1.5

-3.9

PDS5A

PDS5, regulator of cohesion maintenance, TV2

NM_015200.1

2.4

-2.4

-2.1

1.9

MIS12

MIS12, MIND kinetochore complex component, TV2

NM_024039.1

2.4

-1.5

-1.5

-3.4

CDC23

Cell division cycle 23

NM_004661.3

2.4

-1.3

-1.2

1.3

POLS

PAP associated domain containing 7, TV1

NM_006999.3

2.4

-1.8

-1.5

-3.4

CDC25C

Cell division cycle 25C, TV1

NM_001790.3

2.3

1.1

1.1

1.1

BUB1B

BUB1 mitotic checkpoint serine/threonine kinase B

NM_001211.4

2.3

-1.1

-1.4

-2.0

ANAPC11

Anaphase promoting complex subunit 11, TV4

NM_001002246.1

2.3

1.1

1.1

-2.3

CHEK1

Checkpoint kinase 1, TV3

NM_001274.3

2.3

-1.1

-1.4

1.4

ZW10

Zw10 kinetochore protein

NM_004724.2

2.3

-1.4

-1.9

-1.2

SMC2

Structural maintenance of chromosomes 2, TV1

NM_001042550.1

2.3

-1.6

-1.8

-1.3

CUL1

Cullin 1

NM_003592.2

2.3

-1.6

-1.8

-4.5

TUBA1C

Tubulin, alpha 1c

NM_032704.3

2.2

1.0

1.0

2.3

SMC4

Structural maintenance of chromosomes 4, TV2

NM_001002800.1

2.2

1.4

-2.0

1.1

CAPZA2

Capping protein (actin filament) muscle Z-line, alpha 2

NM_006136.2

2.2

-2.0

-1.3

-3.1

PCNA

Proliferating cell nuclear antigen

NM_182649.1

2.1

-1.1

-1.4

1.2

PPP2R5C

Protein phosphatase 2, regulatory subunit B', gamma isoform , TV4

NM_178588.1

2.1

-1.0

-1.2

1.1

SKP1A

S-phase kinase-associated protein 1, TV1

NM_006930.2

2.1

-1.4

-1.8

1.4

TGFBR2

Transforming growth factor, beta receptor II (70/80 kDa), TV1

NM_001024847.1

2.1

-1.5

-1.8

-3.1

CHTF18

CTF18, chromosome transmission fidelity factor 18

NM_022092.1

2.1

-1.1

1.3

1.4

CCNE1

Cyclin E1, TV2

NM_057182.1

2.1

-1.2

-1.1

1.8

YWHAZ

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide

NM_003406.2

2.1

-1.6

-1.6

1.7

CCT8

Chaperonin containing TCP1, subunit 8 (theta)

NM_006585.2

2.1

-1.3

-1.8

-3.7

TUBB4Q

Tubulin, beta polypeptide 4, member Q, pseudogene

NM_020040.3

2.1

-1.0

1.2

1.9

WEE1

WEE1 homolog (S. pombe), TV1

NM_003390.2

2.1

-1.7

-2.2

1.4

RB1

Retinoblastoma 1

NM_000321.2

2.0

-1.4

-1.7

1.8

PPP2R5B

Protein phosphatase 2, regulatory subunit B', beta

NM_006244.2

-2.0

-1.0

1.2

-2.2

CCNA1

Cyclin A1, TV1

NM_003914.2

-2.2

1.2

1.0

1.4

TOB1

Transducer of ERBB2, 1, TV1

NM_005749.2

-2.3

-1.3

-1.4

1.2

LIMK2

LIM domain kinase 2, TV1

NM_001031801.1

-2.3

1.2

1.5

-1.2

MYL5

Myosin, light chain 5, regulatory

NM_002477.1

-2.5

1.3

1.5

-1.1

TP63

Tumor protein p63, TV5

NM_001114981.1

-3.8

-1.1

3.7

-1.1

JUNB

Jun B proto-oncogene

NM_002229.2

-4.3

-1.2

1.6

1.2

PIK3R1

Phosphoinositide-3-kinase, regulatory subunit 1 (alpha), TV1

NM_181523.1

-4.7

-2.1

1.7

2.1

CDKN1A

Cyclin-dependent kinase inhibitor 1A (p21, Cip1)

NM_000389.2

-4.7

1.1

2.5

1.1

RAC1

Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), Rac1b

NM_018890.2

-7.6

-1.0

1.6

1.1

FOS

FBJ murine osteosarcoma viral oncogene homolog

NM_005252.2

-14.9

1.0

2.4

1.4

Apoptosis

      

PAK2

p21 protein (Cdc42/Rac)-activated kinase 2

NM_002577.3

6.0

-2.9

-3.1

-1.3

CDC2

Cyclin-dependent kinase 1, TV1

NM_001786.2

5.2

-1.2

-2.3

1.7

BARD1

BRCA1 associated RING domain 1

NM_000465.1

3.5

-1.7

-2.8

-1.7

MAP2K4

Mitogen-activated protein kinase kinase 4

NM_003010.2

3.3

-2.3

-2.5

-3.1

MAP3K4

Mitogen-activated protein kinase kinase kinase 4, TV1

NM_005922.2

2.6

-1.6

-2.2

-2.5

MAP3K1

Mitogen-activated protein kinase kinase kinase 1, E3 ubiquitin protein ligase

NM_005921.1

2.6

-2.4

-1.6

-3.8

BIRC2

Baculoviral IAP repeat containing 2, TV1

NM_001166.3

2.6

-1.4

-2.1

1.5

SENP2

SUMO1/sentrin/SMT3 specific peptidase 2

NM_021627.2

2.5

-1.7

-1.8

-1.6

RIPK1

Receptor (TNFRSF)-interacting serine-threonine kinase 1

NM_003804.3

2.5

-1.7

-2.1

-4.5

YWHAQ

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide

NM_006826.2

2.5

-1.2

-1.4

-2.1

MAPK9

Mitogen-activated protein kinase 9, TV JNK2-a2

NM_002752.3

2.4

-1.3

-1.5

-2.5

CASP3

Caspase 3, apoptosis-related cysteine peptidase, TV beta

NM_032991.2

2.2

-1.3

-1.7

-4.0

APAF1

Apoptotic peptidase activating factor 1, TV1

NM_013229.2

2.2

-1.5

-1.4

-2.0

BMF

Bcl2 modifying factor, TV2

NM_033503.3

-2.0

1.0

1.1

-1.2

CFLAR

CASP8 and FADD-like apoptosis regulator

NM_003879.3

-2.3

1.2

1.1

-1.9

HSPB1

Heat shock 27 kDa protein 1

NM_001540.2

-2.7

1.2

1.5

-1.1

GADD45B

Growth arrest and DNA-damage-inducible, beta

NM_015675.2

-3.8

1.1

1.8

-1.1

TNFRSF6B

Tumor necrosis factor receptor superfamily, member 6b, decoy, transcript variant M68C

NM_032945.2

-4.3

-1.0

1.2

1.8

Heat Shock

      

HSP90AA1

Heat shock protein 90 kDa, class A member 1

NM_001017963.2

4.0

-1.4

-1.7

1.1

CARHSP1

Calcium regulated heat stable protein 1, 24 kDa

NM_001042476.1

3.2

1.0

1.1

-1.1

HSPA12A

Heat shock protein 70 kDa 12A

NM_025015.2

2.3

-1.2

-1.7

1.4

HSPB1

Heat shock protein 27 kDa protein 1

NM_001540.2

-2.7

1.2

1.5

-1.1

HSPBL2

Heat shock 27 kDa protein 1 pseudogene 1

NR_024392.1

-3.2

1.2

1.6

-1.0

HSPA6

Heat shock 70 kDa protein 6

NM_002155.3

-5.3

1.2

1.1

1.3

HSPA7

Heat shock 70 kDa protein 7

NR_024151.1

-4.0

1.2

1.4

1.2

Identification of hyperthermia induced genes that differentiate the heat shock response of mammary epithelial cells from that of breast cancer cells

Our previous analysis compared the hyperthermic response of each individual cell line to its transcriptional expression baseline at the normal growth temperature (C vs H and C’ vs H’). Though this analysis provides us with information on how each individual cell line responds to hyperthermia relative to its normal growth temperature, it does not provide absolute comparisons of the transcriptome response of breast cancer cells relative to mammary epithelial cells following the elevated temperature. To better understand what provides breast cancer cells the selective disadvantage over mammary epithelial cells in response to hyperthermia we must identify those genes that are differentially expressed in breast cancer cell lines following hyperthermia from those of the mammary epithelial cell line following hyperthermia. To perform this analysis, we directly compared the gene expression changes that occurred for H’ vs H and identified genes whose expression was truly distinct between the breast cancer and mammary epithelial cell lines following hyperthermia.

When comparing H’ (MCF7) vs H we identified 2708 genes whose expression was distinct at statistically significant levels (≥2 fold, p < 0.05). H’ vs H comparisons of the MDA231 and MDA468 lines yielded 919 and 750 significant gene expression changes, respectively. Heatmap analysis indicated a strong trend in the gene expression profiles between each of the three breast cancer lines following hyperthermia (Figure 3A). Using a Venn diagram that strictly eliminated any genes with less than a 2-fold expression change (p < 0.05), we compared the gene expression alterations that were uniquely shared between all three cancer lines (H’) relative to the mammary epithelial line (H) (Figure 3B). This interpretation uncovered 393 genes whose 2-fold or greater changes in gene expression were differentially expressed in common amongst the three breast cancer lines following mild hyperthermic shock when compared to MCF10A cells following the same treatment (Table 3). These are the core genes that differentiate the hyperthermic response of breast cancer cells from that of mammary epithelial cells. In these data potentially lay the mechanism that may help define how mild hyperthermia preferentially selects against tumor cells.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-81/MediaObjects/12885_2013_Article_4319_Fig3_HTML.jpg
Figure 3

Identification of genes that differentiate the transcriptional response of breast cancer cells following fever range hyperthermia. (A) Heatmap depicting the two-fold or greater changes in gene expression (p < 0.05) occurring in at least one of the three breast cancer cell lines (MCF7, MDA231, MDA468) relative to the mammary epithelial cells in the H’ vs H comparison (red?=?overexpressed, green?=?underexpressed). (B) Venn diagram illustrating common and unique 2-fold or greater gene expression changes (p < 0.05) between each of the breast cancer cell lines relative to the mammary epithelial cells in the H’ vs H comparison.

Table 3

List of genes that statistically distinguish the hyperthermic response of three breast cancer lines from the mammary epithelial cells in the H’ vs H analysis

Gene Symbol

Gene Name

Accession Number

MCF-7

MDA-231

MDA-468

RN7SK

RNA, 7SK small nuclear

NR_001445.1

68.5

64.5

73.5

RN5S9

RNA, 5S ribosomal 9

NR_023371.1

54.5

64.7

64.9

RNU1-3

RNA, U1 small nuclear 3

NR_004408.1

44.2

27.9

30.3

RNU1G2

RNA, U1 small nuclear 4

NR_004426.1

42.5

26.4

28.5

KIAA1666

RIMS binding protein 3

XM_942124.2

38.5

36.1

41.6

RNU1-5

RNA, U1 small nuclear 5

NR_004400.1

36.5

25.1

28.1

SNORD3D

Small nucleolar RNA, C/D box 3D

NR_006882.1

34.8

43.3

47.2

RNU1A3

RNA, U1 small nuclear 1

NR_004430.1

30.2

19.9

20.3

SNORD3A

Small nucleolar RNA, C/D box 3A

NR_006880.1

30.1

42.6

43.8

SNORD3C

Small nucleolar RNA, C/D box 3C

NR_006881.1

28.4

30.9

34.7

RNY5

RNA, Ro-associated Y5

NR_001571.2

27.8

4.6

4.3

LOC100008589

RNA28S5 RNA, 28S ribosomal 5

NR_003287.1

27.2

28.9

30.9

LOC100132564

Hypothetical Protein LOC100132564

XM_001713808.1

26.7

26.5

27.3

LOC100132394

Hypothetical Protein

XP_001713861.1

23.8

23.1

22.9

RNU4-1

RNA, U4 small nuclear 1

NR_003925.1

18.5

11.6

13.1

RNU6ATAC

RNA, U6atac small nuclear (U12-dependent splicing)

NR_023344.1

16.3

11.8

12.4

LOC100134364

Hypothetical Protein LOC100134364

XM_001713810.1

15.9

15.5

17.2

HIST2H2AA3

Histone cluster 2, H2aa3

NM_003516.2

15.3

3.2

3.1

HIST2H2AA4

Histone cluster 2, H2aa4

NM_001040874.1

13.4

3.1

2.8

MIR1974

MicroRNA 1974

NR_031738.1

12.8

24.1

24.5

RMRP

RNA component of mitochondrial RNA processing endoribonuclease

NR_003051.2

12.1

14.1

15.6

RNA18S5

RNA18S5 RNA, 18S ribosomal 5

NR_003286.1

11.4

11.9

12.6

RNU4-2

RNA, U4 small nuclear 2

NR_003137.2

10.7

5.7

6.3

SCARNA20

Small Cajal body-specific RNA 20

NR_002999.2

7.7

3.2

3.1

LOC441763

Hypothetical Protein LOC441763

XM_930284.1

7.4

5.9

7.2

LOC728688

Ubiquitin-like, containing PHD and RING finger domains

XM_001724542.1

6.6

6.1

7.3

VTRNA1-1

Vault RNA 1-1

NR_026703.1

6.5

4.4

4.1

RNU6-1

RNA, U6 small nuclear 1

NR_004394.1

6.3

6.9

7.3

RNU6-15

RNA, U6 small nuclear 15

NR_028372.1

6.3

6.2

7.1

ALB

Albumin

NM_000477.3

4.9

5.3

5.5

HIST1H4H

Histone cluster 1, H4h

NM_003543.3

4.9

2.9

2.9

HIST2H4A

Histone cluster 2, H4a

NM_003548.2

4.6

5.1

4.7

TRK1

Transfer RNA lysine 1 (anticodon UUU)

NR_001449.1

4.6

3.5

3.8

SNORD46

Small nucleolar RNA, C/D box 46

NR_000024.2

4.2

3.9

3.8

RNU4ATAC

RNA, U4atac small nuclear (U12-dependent splicing)

NR_023343.1

4.2

3.7

3.9

KREMEN2

Kringle containing transmembrane protein 2

NM_145348.1

4.1

2.9

2.8

RNU11

RNA, U11 small nuclear

NR_004407.1

3.8

2.8

2.9

SNORA57

Small nucleolar RNA, H/ACA box 57

NR_004390.1

3.8

4.9

5.2

RPPH1

Ribonuclease P RNA component H1

NR_002312.1

3.7

2.7

3.1

SCARNA13

Small Cajal body-specific RNA 13

NR_003002.1

3.7

4.3

4.9

RPL12P6

Ribosomal protein L12 pseudogene 6

XR_016704.2

3.5

2.4

4.1

LOC389787

Tumor protein, translationally-controlled 1 pseudogene

XM_497072.2

3.4

3.1

2.1

RPL10L

Ribosomal protein L10-like

NM_080746.2

3.3

3.6

3.7

SNORA7B

Small nucleolar RNA, H/ACA box 7B

NR_002992.2

3.2

4.8

5.1

LOC100132673

Ribosomal protein S2 pseudogene 28

XR_039018.1

3.1

2.3

2.6

RNY4

RNA, Ro-associated Y4

NR_004393.1

3.1

5.5

4.7

HOXB6

Homeobox B6

NM_018952.4

2.9

4.2

4.1

HIST1H4K

Histone cluster 1, H4k

NM_003541.2

2.9

3.2

3.3

SNORA12

Small nucleolar RNA, H/ACA box 12

NR_002954.1

2.9

2.4

2.3

LOC643031

MT-ND5 pseudogene 10

XM_926402.1

2.8

2.9

3.8

HIST2H4B

Histone cluster 2, H4b

NM_001034077.4

2.7

2.8

2.9

HIST2H3D

Histone cluster 2, H3d

NM_001123375.1

2.3

3.7

3.9

HIST1H2AC

Histone cluster 1, H2ac

NM_003512.3

2.3

2.9

2.7

EGR1

Early growth response 1

NM_001964.2

2.3

2.8

2.4

SNORA63

Small nucleolar RNA, H/ACA box 63

NR_002586.1

2.3

7.8

7.3

SNORD13

Small nucleolar RNA, C/D box 13

NR_003041.1

2.3

5.6

5.7

RNY1

RNA, Ro-associated Y1

NR_004391.1

2.1

4.4

4.1

HIST1H2AM

Histone cluster 1, H2am

NM_003514.2

2.1

3.1

3.5

MARCH7

Membrane-associated ring finger 7, E3 ubiquitin protein ligase

NM_022826.2

-2.0

-2.3

-2.3

GPBP1

GC-rich promoter binding protein 1

NM_022913.1

-2.0

-3.4

-2.9

RAB11FIP1

RAB11 family interacting protein 1 (class I)

NM_001002814.1

-2.0

-2.0

-2.8

CEBPG

CCAAT/enhancer binding protein (C/EBP), gamma

NM_001806.2

-2.0

-2.0

-2.5

ZWILCH

Zwilch kinetochore protein

NR_003105.1

-2.0

-2.7

-2.6

DDX18

DEAD (Asp-Glu-Ala-Asp) box polypeptide 18

NM_006773.3

-2.0

-2.6

-2.7

PTP4A1

Protein tyrosine phosphatase type IVA, member 1

NM_003463.3

-2.0

-2.9

-2.4

DDX3X

DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked

NM_001356.3

-2.0

-2.6

-2.3

CTCF

CCCTC-binding factor (zinc finger protein)

NM_006565.2

-2.0

-2.6

-2.3

SKAP2

src kinase associated phosphoprotein 2

NM_003930.3

-2.0

-2.3

-2.2

ANO6

Anoctamin 6

NM_001025356.1

-2.0

-2.3

-2.3

PPAT

Phosphoribosyl pyrophosphate amidotransferase

NM_002703.3

-2.0

-2.1

-2.3

USP34

Ubiquitin specific peptidase 34

NM_014709.3

-2.0

-2.1

-2.0

LHFPL2

Lipoma HMGIC fusion partner-like 2

NM_005779.1

-2.1

-2.1

-2.1

KIAA1147

KIAA1147

NM_001080392.1

-2.1

-2.1

-2.3

KIAA2010

SMEK homolog 1, suppressor of mek1 (Dictyostelium)

NM_032560.3

-2.1

-3.6

-3.0

DYRK1A

Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A

NM_130438.1

-2.1

-2.5

-2.3

RBBP8

Retinoblastoma binding protein 8

NM_203291.1

-2.1

-2.6

-2.4

ANKIB1

Ankyrin repeat and IBR domain containing 1

NM_019004.1

-2.1

-2.5

-2.5

C12orf32

RAD9-HUS1-RAD1 interacting nuclear orphan 1

NM_031465.2

-2.1

-2.1

-2.1

FNIP1

Folliculin interacting protein 1

NM_001008738.2

-2.1

-2.0

-2.1

NAE1

NEDD8 activating enzyme E1 subunit 1

NM_001018160.1

-2.2

-2.0

-2.3

RSBN1

Round spermatid basic protein 1

NM_018364.3

-2.2

-2.0

-2.3

SUPT16H

Suppressor of Ty 16 homolog (S. cerevisiae)

NM_007192.2

-2.2

-2.1

-2.2

TMED10

Transmembrane emp24-like trafficking protein 10 (yeast)

NM_006827.5

-2.2

-3.6

-2.9

PPP1CB

Protein phosphatase 1, catalytic subunit, beta isozyme

NM_206876.1

-2.2

-2.4

-2.7

ARL6IP1

ADP-ribosylation factor-like 6 interacting protein 1

NM_015161.1

-2.2

-2.8

-2.7

ASNSD1

Asparagine synthetase domain containing 1

NM_019048.1

-2.2

-2.9

-2.5

NRBF2P4

Nuclear receptor binding factor 2 pseudogene 4

XM_001127763.1

-2.2

-2.6

-2.4

TTC37

Tetratricopeptide repeat domain 37

NM_014639.2

-2.2

-2.6

-2.2

DICER1

Dicer 1, ribonuclease type III

NM_030621.2

-2.2

-2.6

-2.3

UBE2G1

Ubiquitin-conjugating enzyme E2G 1

NM_003342.4

-2.2

-2.5

-2.4

LRPPRC

Leucine-rich pentatricopeptide repeat containing

NM_133259.2

-2.2

-2.4

-2.2

OTUD4

OTU domain containing 4

NM_199324.1

-2.2

-2.3

-2.2

FUBP3

Far upstream element (FUSE) binding protein 3

NM_003934.1

-2.2

-2.3

-2.2

FAM175B

Family with sequence similarity 175, member B

NM_032182.3

-2.2

-2.1

-2.1

DDX50

DEAD (Asp-Glu-Ala-Asp) box polypeptide 50

NM_024045.1

-2.2

-2.0

-2.1

PIGA

Phosphatidylinositol glycan anchor biosynthesis, class A

NM_020473.2

-2.2

-2.0

-2.2

FAM168B

Family with sequence similarity 168, member B

NM_001009993.2

-2.3

-2.0

-2.0

FBXO11

F-box protein 11

NM_025133.3

-2.3

-2.1

-2.5

SLC25A24

Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 24

NM_013386.3

-2.3

-3.4

-2.5

NCKAP1

NCK-associated protein 1

NM_013436.3

-2.3

-3.3

-2.7

FBXW11

F-box and WD repeat domain containing 11

NM_033645.2

-2.3

-2.5

-2.7

SLC30A9

Solute carrier family 30 (zinc transporter), member 9

NM_006345.3

-2.3

-2.0

-2.7

ATAD2

ATPase family, AAA domain containing 2

NM_014109.2

-2.3

-2.8

-2.0

CSNK1A1

Casein kinase 1, alpha 1

NM_001025105.1

-2.3

-2.5

-2.2

CHUK

Conserved helix-loop-helix ubiquitous kinase

NM_001278.3

-2.3

-2.2

-2.3

ATP6V1C1

ATPase, H?+?transporting, lysosomal 42 kDa, V1 subunit C1

NM_001695.4

-2.3

-2.1

-2.4

CUL2

Cullin 2

NM_003591.2

-2.3

-2.2

-2.1

BRMS1L

Breast cancer metastasis-suppressor 1-like

NM_032352.3

-2.3

-2.1

-2.0

UBQLN1

Ubiquilin 1

NM_013438.3

-2.3

-2.1

-2.1

OPA1

Optic atrophy 1 (autosomal dominant)

NM_015560.1

-2.4

-2.7

-2.6

EFR3A

EFR3 homolog A (S. cerevisiae)

NM_015137.3

-2.4

-2.3

-3.2

BAT2D1

Proline-rich coiled-coil 2C

NM_015172.3

-2.4

-2.5

-2.1

OSBPL9

Oxysterol binding protein-like 9

NM_148906.1

-2.4

-2.6

-2.5

CDR2

Cerebellar degeneration-related protein 2, 62 kDa

NM_001802.1

-2.4

-2.3

-2.2

KPNA4

Karyopherin alpha 4 (importin alpha 3)

NM_002268.3

-2.4

-2.2

-2.4

ARL1

ADP-ribosylation factor-like 1

NM_001177.3

-2.4

-2.2

-2.3

DHX32

DEAH (Asp-Glu-Ala-His) box polypeptide 32

NM_018180.2

-2.4

-2.1

-2.3

RIOK3

RIO kinase 3

NM_003831.3

-2.4

-2.0

-2.1

C5orf51

Chromosome 5 open reading frame 51

NM_175921.4

-2.5

-2.8

-2.6

TJP1

Tight junction protein 1

NM_003257.3

-2.5

-2.4

-2.2

CSNK1G3

Casein kinase 1, gamma 3

NM_001031812.2

-2.5

-2.4

-2.3

PREI3

MOB family member 4, phocein

NM_199482.1

-2.5

-2.4

-2.4

KIAA0494

EF-hand calcium binding domain 14

NM_014774.1

-2.5

-2.3

-2.4

PRKRIR

Protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 repressor)

NM_004705.2

-2.5

-2.7

-3.0

LOC644363

LOC644363

XR_016912.2

-2.5

-2.6

-2.9

DCP2

DCP2 decapping enzyme homolog (S. cerevisiae)

NM_152624.4

-2.5

-2.5

-3.0

ROD1

Polypyrimidine tract binding protein 3

NM_005156.4

-2.5

-2.9

-3.2

CRK

v-crk sarcoma virus CT10 oncogene homolog (avian)

NM_016823.2

-2.5

-3.3

-2.8

PRKAR1A

Protein kinase, cAMP-dependent, regulatory, type I, alpha

NM_002734.3

-2.5

-2.6

-2.4

GPSM2

G-protein signaling modulator 2

NM_013296.3

-2.5

-2.2

-2.3

API5

Apoptosis inhibitor 5

NM_006595.2

-2.5

-2.1

-2.5

CRKL

v-crk sarcoma virus CT10 oncogene homolog (avian)-like

NM_005207.2

-2.5

-2.0

-2.6

CBL

Cbl proto-oncogene, E3 ubiquitin protein ligase

NM_005188.2

-2.5

-2.0

-2.2

SMG1

smg-1 homolog, phosphatidylinositol 3-kinase-related kinase interferon

NM_015092.3

-2.6

-2.9

-2.6

IRF2BP2

Regulatory factor 2 binding protein 2

NM_182972.2

-2.6

-2.7

-2.7

CNOT6

CCR4-NOT transcription complex, subunit 6

NM_015455.3

-2.6

-2.7

-2.6

THUMPD1

THUMP domain containing 1

NM_017736.3

-2.6

-2.6

-2.5

BMI1

BMI1 polycomb ring finger oncogene

NM_005180.5

-2.6

-2.4

-2.7

CDC2L6

Cyclin-dependent kinase 19

NM_015076.3

-2.6

-2.4

-2.8

TULP4

Tubby like protein 4

NM_001007466.1

-2.6

-2.3

-2.6

LARP4B

La ribonucleoprotein domain family, member 4B

NM_015155.1

-2.6

-2.3

-2.3

HECTD1

HECT domain containing E3 ubiquitin protein ligase 1

NM_015382.1

-2.6

-2.6

-2.4

CPSF2

Cleavage and polyadenylation specific factor 2, 100 kDa

NM_017437.1

-2.6

-2.8

-2.6

PDS5A

PDS5, regulator of cohesion maintenance, homolog A (S. cerevisiae)

NM_015200.1

-2.6

-2.6

-2.9

RIPK1

Receptor (TNFRSF)-interacting serine-threonine kinase 1

NM_003804.3

-2.6

-2.1

-2.4

DCAF6

DDB1 and CUL4 associated factor 6

NM_001017977.1

-2.6

-2.1

-2.4

KIAA1429

KIAA1429

NM_015496.3

-2.6

-2.0

-2.3

RUNX1

Runt-related transcription factor 1

NM_001754.3

-2.6

-2.1

-2.2

CEP135

Centrosomal protein 135 kDa

NM_025009.3

-2.6

-2.0

-2.2

MBNL1

Muscleblind-like splicing regulator 1

NM_207296.1

-2.6

-2.5

-2.2

RBPJ

Recombination signal binding protein for immunoglobulin kappa J region

NM_203284.1

-2.6

-2.4

-2.0

USP16

Ubiquitin specific peptidase 16

NM_006447.2

-2.6

-2.2

-2.1

TOMM20

Translocase of outer mitochondrial membrane 20 homolog (yeast)

NM_014765.1

-2.7

-2.3

-2.5

MTX3

Metaxin 3

NM_001010891.3

-2.7

-2.7

-2.6

RAPH1

Ras association (RalGDS/AF-6) and pleckstrin homology domains 1

NM_213589.1

-2.7

-2.6

-2.5

PHF20L1

PHD finger protein 20-like 1

NM_016018.4

-2.7

-2.6

-2.7

UBP1

Upstream binding protein 1 (LBP-1a)

NM_014517.3

-2.7

-2.5

-2.6

GBE1

Glucan (1,4-alpha-), branching enzyme 1

NM_000158.2

-2.7

-2.4

-2.3

CUL4B

Cullin 4B

NM_001079872.1

-2.7

-2.4

-2.4

PAPOLA

Poly(A) polymerase alpha

NM_001037281.1

-2.7

-2.4

-2.9

RNMT

RNA (guanine-7-) methyltransferase

NM_003799.1

-2.7

-2.1

-2.4

FBXO34

F-box protein 34

NM_017943.2

-2.7

-2.3

-2.1

DOCK7

Dedicator of cytokinesis 7

NM_033407.2

-2.7

-2.3

-2.1

BTBD3

BTB (POZ) domain containing 3

NM_014962.2

-2.7

-2.2

-2.1

C6orf130

O-acyl-ADP-ribose deacylase 1

NM_145063.2

-2.8

-2.3

-2.1

MRPL35

Mitochondrial ribosomal protein L35

NM_016622.2

-2.8

-2.2

-2.2

PUM2

Pumilio homolog 2 (Drosophila)

NM_015317.1

-2.8

-2.8

-2.8

NUDT21

Nudix (nucleoside diphosphate linked moiety X)-type motif 21

NM_007006.2

-2.8

-2.7

-2.3

ICK

Intestinal cell (MAK-like) kinase

NM_016513.3

-2.8

-2.7

-2.5

RBM17

RNA binding motif protein 17

NM_032905.3

-2.8

-2.4

-2.1

RMI1

RMI1, RecQ mediated genome instability 1, homolog (S. cerevisiae)

NM_024945.2

-2.9

-2.4

-2.1

MAP2K4

Mitogen-activated protein kinase kinase 4

NM_003010.2

-2.9

-2.8

-2.8

G3BP2

GTPase activating protein (SH3 domain) binding protein 2

NM_203504.1

-2.9

-2.5

-2.5

NAMPT

Nicotinamide phosphoribosyltransferase

NM_005746.2

-2.9

-2.7

-2.1

BEND7

BEN domain containing 7

NM_001100912.1

-2.9

-2.2

-2.1

FEZ2

Fasciculation and elongation protein zeta 2 (zygin II)

NM_005102.2

-3.0

-2.2

-2.2

ARL4A

ADP-ribosylation factor-like 4A

NM_001037164.1

-3.0

-2.4

-2.2

SOCS4

Suppressor of cytokine signaling 4

NM_080867.2

-3.0

-3.0

-2.6

STAG2

Stromal antigen 2

NM_001042750.1

-3.0

-2.9

-2.5

C14orf32

Mitogen-activated protein kinase 1 interacting protein 1-like

NM_144578.2

-3.0

-2.9

-2.9

PPP3CB

Protein phosphatase 3, catalytic subunit, beta isozyme

NM_021132.1

-3.0

-2.4

-2.6

RBM25

RNA binding motif protein 25

NM_021239.1

-3.0

-2.4

-2.5

ERI1

Exoribonuclease 1

NM_153332.3

-3.0

-2.0

-2.3

NRD1

Nardilysin (N-arginine dibasic convertase)

NM_002525.1

-3.0

-2.4

-2.1

E2F3

E2F transcription factor 3

NM_001949.2

-3.0

-2.0

-2.0

ANKRD28

Ankyrin repeat domain 28

NM_015199.2

-3.1

-2.3

-2.2

ZAK

Sterile alpha motif and leucine zipper containing kinase AZK

NM_133646.2

-3.1

-3.1

-3.4

HNRPR

Heterogeneous nuclear ribonucleoprotein R

NM_005826.2

-3.1

-2.9

-2.1

TMEM123

Transmembrane protein 123

NM_052932.2

-3.1

-2.8

-2.3

FAM178A

Family with sequence similarity 178, member A

NM_018121.3

-3.1

-2.7

-2.6

EML4

Echinoderm microtubule associated protein like 4

NM_019063.2

-3.1

-2.6

-2.6

FOXJ3

Forkhead box J3

NM_014947.3

-3.1

-2.5

-2.5

NT5DC3

5'-nucleotidase domain containing 3

NM_016575.1

-3.1

-2.3

-2.6

LPP

LIM domain containing preferred translocation partner in lipoma

NM_005578.2

-3.1

-2.2

-3.5

RND3

Rho family GTPase 3

NM_005168.3

-3.1

-2.0

-2.2

WDR36

WD repeat domain 36

NM_139281.2

-3.1

-2.0

-2.2

CDCA1

NUF2, NDC80 kinetochore complex component

NM_145697.1

-3.1

-2.5

-2.0

PRSS23

Protease, serine, 23

NM_007173.4

-3.2

-2.3

-2.0

ERCC6L

Excision repair cross-complementing rodent repair deficiency, complementation group 6-like

NM_017669.2

-3.2

-2.3

-2.0

FAM122B

Family with sequence similarity 122B

NM_032448.1

-3.3

-2.5

-2.0

CKAP5

Cytoskeleton associated protein 5

NM_001008938.1

-3.3

-2.2

-2.1

CGGBP1

CGG triplet repeat binding protein 1

NM_001008390.1

-3.3

-2.8

-3.5

TBL1XR1

Transducin (beta)-like 1 X-linked receptor 1

NM_024665.3

-3.3

-3.1

-2.7

LOC644799

LOC644799

XM_934554.1

-3.3

-3.0

-2.5

DCK

Deoxycytidine kinase

NM_000788.1

-3.3

-2.8

-2.2

SERBP1

SERPINE1 mRNA binding protein 1

NM_030666.2

-3.3

-2.1

-3.3

PPP1CC

Protein phosphatase 1, catalytic subunit, gamma isozyme

NM_002710.1

-3.3

-2.2

-2.1

KIF5B

Kinesin family member 5B

NM_004521.1

-3.4

-4.1

-3.3

AFF4

AF4/FMR2 family, member 4

NM_014423.3

-3.4

-3.6

-3.5

MPP5

Membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5)

NM_022474.2

-3.4

-3.1

-2.7

IPO5

Importin 5

NM_002271.4

-3.4

-2.6

-2.2

HNRNPR

Heterogeneous nuclear ribonucleoprotein R

NM_005826.3

-3.4

-2.6

-2.3

CP110

Centriolar coiled coil protein 110 kDa

NM_014711.3

-3.4

-2.5

-2.5

FEM1C

fem-1 homolog c (C. elegans)

NM_020177.2

-3.4

-2.4

-2.5

PHTF1

Putative homeodomain transcription factor 1

NM_006608.1

-3.4

-2.3

-2.1

RAD51AP1

RAD51 associated protein 1

NM_006479.3

-3.4

-2.2

-2.0

MAPRE1

Microtubule-associated protein, RP/EB family, member 1

NM_012325.1

-3.4

-2.1

-2.1

TMPO

Thymopoietin

NM_003276.1

-3.5

-3.1

-2.3

LACTB

Lactamase, beta

NM_032857.2

-3.5

-2.3

-2.3

DDX46

DEAD (Asp-Glu-Ala-Asp) box polypeptide 46

NM_014829.2

-3.5

-2.3

-2.4

SPEN

Spen homolog, transcriptional regulator (Drosophila)

NM_015001.2

-3.5

-2.0

-2.7

TMEM19

Transmembrane protein 19

NM_018279.3

-3.5

-2.1

-2.0

CBFB

Core-binding factor, beta subunit

NM_001755.2

-3.5

-2.2

-2.2

IPO8

Importin 8

NM_006390.2

-3.5

-2.2

-2.1

WT1

Wilms tumor 1

NM_024426.3

-3.5

-2.4

-2.1

CKAP2

Cytoskeleton associated protein 2

NM_001098525.1

-3.6

-3.0

-2.4

WEE1

WEE1 homolog (S. pombe)

NM_003390.2

-3.6

-2.8

-2.7

PDCD6IP

Programmed cell death 6 interacting protein

NM_013374.3

-3.6

-2.7

-2.3

ZNF788

Zinc finger family member 788

XR_041527.1

-3.6

-2.6

-2.3

RAP2A

RAP2A, member of RAS oncogene family

NM_021033.5

-3.6

-2.3

-2.7

MGEA5

Meningioma expressed antigen 5 (hyaluronidase)

NM_012215.2

-3.6

-2.1

-2.7

UBE3A

Ubiquitin protein ligase E3A

NM_000462.2

-3.6

-2.0

-2.8

PLK4

Polo-like kinase 4

NM_014264.3

-3.7

-3.8

-3.2

RP2

Retinitis pigmentosa 2 (X-linked recessive)

NM_006915.1

-3.7

-2.9

-2.5

SETD2

SET domain containing 2

NM_014159.4

-3.7

-2.7

-2.8

KLHL5

Kelch-like family member 5

NM_001007075.1

-3.7

-2.4

-2.6

KBTBD2

Kelch repeat and BTB (POZ) domain containing 2

NM_015483.1

-3.7

-3.7

-3.1

USP9X

Ubiquitin specific peptidase 9, X-linked

NM_001039591.2

-3.8

-2.8

-2.4

RAB23

RAB23, member RAS oncogene family

NM_016277.3

-3.8

-2.6

-2.5

DR1

Down-regulator of transcription 1, TBP-binding (negative cofactor 2)

NM_001938.2

-3.8

-2.3

-2.2

RAB8B

RAB8B, member RAS oncogene family

NM_016530.2

-3.8

-2.3

-2.5

ZNF451

Zinc finger protein 451

NM_001031623.2

-3.8

-2.2

-2.4

ZZZ3

Zinc finger, ZZ-type containing 3

NM_015534.4

-3.9

-3.1

-3.3

CDC2

Cyclin-dependent kinase 1

NM_001786.2

-4.0

-2.9

-2.3

ZFP106

Zinc finger protein 106

NM_022473.1

-4.0

-3.0

-2.5

CAB39

Calcium binding protein 39

NM_016289.2

-4.0

-2.0

-3.1

TNPO1

Transportin 1

NM_153188.2

-4.0

-2.9

-2.5

MAP3K4

Mitogen-activated protein kinase kinase kinase 4

NM_005922.2

-4.1

-2.3

-2.7

ECT2

Epithelial cell transforming sequence 2 oncogene

NM_018098.4

-4.1

-2.3

-2.0

TMED5

Transmembrane emp24 protein transport domain containing 5

NM_016040.3

-4.1

-2.3

-2.1

SEH1L

SEH1-like (S. cerevisiae)

NM_001013437.1

-4.1

-2.7

-2.5

NCAPG2

Non-SMC condensin II complex, subunit G2

NM_017760.5

-4.2

-2.7

-2.2

USP1

Ubiquitin specific peptidase 1

NM_001017416.1

-4.2

-2.3

-2.4

OXSR1

Oxidative-stress responsive 1

NM_005109.2

-4.2

-2.3

-2.4

PTPN12

Protein tyrosine phosphatase, non-receptor type 12

NM_002835.2

-4.2

-2.8

-2.8

CMPK1

Cytidine monophosphate (UMP-CMP) kinase 1, cytosolic

NM_016308.1

-4.2

-2.5

-2.4

PAFAH1B1

Platelet-activating factor acetylhydrolase 1b, regulatory subunit 1 (45 kDa)

NM_000430.2

-4.3

-3.8

-3.1

PURB

Purine-rich element binding protein B

NM_033224.3

-4.3

-2.5

-3.5

STK4

Serine/threonine kinase 4

NM_006282.2

-4.3

-2.3

-3.3

KATNAL1

Katanin p60 subunit A-like 1

NM_001014380.1

-4.3

-3.1

-2.9

NIN

Ninein (GSK3B interacting protein)

NM_020921.3

-4.3

-2.7

-2.4

LOC283267

Long intergenic non-protein coding RNA 294

NR_015451.1

-4.3

-2.6

-2.6

CCNB1

Cyclin B1

NM_031966.2

-4.3

-2.4

-2.2

YAP1

Yes-associated protein 1

NM_006106.2

-4.3

-2.0

-2.5

XPO1

Exportin 1 (CRM1 homolog, yeast)

NM_003400.3

-4.4

-2.9

-2.6

PTPN11

Protein tyrosine phosphatase, non-receptor type 11

NM_002834.3

-4.4

-2.4

-2.5

PHF3

PHD finger protein 3

NM_015153.1

-4.4

-2.2

-2.6

VMA21

Vacuolar H?+?-ATPase homolog (S. cerevisiae)

NM_001017980.2

-4.4

-2.2

-2.3

CHST15

Carbohydrate sulfotransferase 15

NM_015892.2

-4.4

-2.0

-2.5

RUNX2

Runt-related transcription factor 2

NM_001024630.2

-4.5

-2.7

-2.4

KIF14

Kinesin family member 14

NM_014875.1

-4.6

-3.4

-2.2

EHBP1

EH domain binding protein 1

NM_015252.2

-4.6

-2.7

-2.6

NEK2

NIMA-related kinase 2

NM_002497.2

-4.6

-2.7

-2.6

STK38

Serine/threonine kinase 38

NM_007271.2

-4.7

-3.2

-2.4

ZNF22

Zinc finger protein 22

NM_006963.3

-4.7

-3.2

-2.8

SPRY4

Sprouty homolog 4 (Drosophila)

NM_030964.2

-4.7

-2.0

-2.1

GMFB

Glia maturation factor, beta

NM_004124.2

-4.8

-2.2

-3.2

GCNT1

Glucosaminyl (N-acetyl) transferase 1, core 2

NM_001097635.1

-4.8

-3.1

-2.4

HERC4

HECT and RLD domain containing E3 ubiquitin protein ligase 4

NM_022079.2

-4.8

-3.1

-2.6

PPP4R1

Protein phosphatase 4, regulatory subunit 1

NM_005134.2

-4.8

-2.1

-2.6

SMAD5

SMAD family member 5

NM_005903.5

-4.9

-2.2

-3.2

GNG12

guanine nucleotide binding protein (G protein), gamma 12

NM_018841.4

-4.9

-2.8

-2.1

SMARCA1

SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1

NM_003069.2

-4.9

-2.8

-2.3

DEK

DEK oncogene

NM_003472.2

-4.9

-2.5

-2.4

FAM107B

Family with sequence similarity 107, member B

NM_031453.2

-5.1

-3.1

-2.1

SUZ12

Suppressor of zeste 12 homolog (Drosophila)

NM_015355.1

-5.1

-2.8

-2.7

OSBPL3

Oxysterol binding protein-like 3

NM_145322.1

-5.1

-2.7

-2.7

UBE3C

Ubiquitin protein ligase E3C

NM_014671.1

-5.1

-2.7

-2.7

HSDL2

Hydroxysteroid dehydrogenase like 2

NM_032303.3

-5.1

-2.5

-2.5

C14orf106

MIS18 binding protein 1

NM_018353.3

-5.1

-2.3

-2.3

MBP

Myelin basic protein

NM_001025100.1

-5.1

-2.0

-2.3

GPAM

Glycerol-3-phosphate acyltransferase, mitochondrial

NM_020918.3

-5.2

-3.1

-2.7

RASA1

RAS p21 protein activator (GTPase activating protein) 1

NM_002890.1

-5.2

-2.9

-3.1

KIF11

Kinesin family member 11

NM_004523.2

-5.2

-3.3

-3.1

FBXO5

F-box protein 5

NM_012177.2

-5.2

-3.1

-2.3

CENPE

Centromere protein E, 312 kDa

NM_001813.2

-5.2

-2.6

-2.3

PAK2

p21 protein (Cdc42/Rac)-activated kinase 2

NM_002577.3

-5.3

-3.3

-3.6

IL7R

Interleukin 7 receptor

XM_937367.1

-5.3

-2.8

-2.2

ENC1

Ectodermal-neural cortex 1 (with BTB domain)

NM_003633.1

-5.3

-2.6

-2.3

SOX9

SRY (sex determining region Y)-box 9

NM_000346.2

-5.3

-2.5

-2.3

ASXL1

Additional sex combs like 1 (Drosophila) (ASXL1), TV1

NM_015338.4

-5.3

-2.0

-2.0

C10orf6

Family with sequence similarity 178, member A

NM_018121.2

-5.5

-3.2

-3.4

CEP55

Centrosomal protein 55 kDa

NM_018131.3

-5.6

-3.4

-2.6

NMT2

N-myristoyltransferase 2

NM_004808.2

-5.6

-2.4

-2.0

PPPDE1

Desumoylating isopeptidase 2

NM_016076.3

-5.8

-2.4

-2.9

TGFBR2

Transforming growth factor, beta receptor II (70/80 kDa)

NM_001024847.1

-6.0

-2.1

-2.2

MID1

Midline 1 (Opitz/BBB syndrome)

NM_033290.2

-6.2

-2.9

-2.1

FNDC3B

Fibronectin type III domain containing 3B

NM_001135095.1

-6.2

-2.8

-2.6

BIRC2

Baculoviral IAP repeat containing 2

NM_001166.3

-6.2

-2.9

-2.7

FAM3C

Family with sequence similarity 3, member C

NM_001040020.1

-6.2

-2.4

-2.0

KIF23

Kinesin family member 23

NM_004856.4

-6.3

-3.4

-2.7

CLIC4

Chloride intracellular channel 4

NM_013943.1

-6.3

-3.4

-2.6

PDGFC

Platelet derived growth factor C

NM_016205.1

-6.4

-2.4

-2.0

PRKCA

Protein kinase C, alpha

NM_002737.2

-6.7

-2.5

-2.2

NCAPG

Non-SMC condensin I complex, subunit G

NM_022346.3

-7.1

-2.6

-2.1

CENPF

Centromere protein F, 350/400 kDa

NM_016343.3

-7.4

-2.9

-2.7

GCNT2

Glucosaminyl (N-acetyl) transferase 2, I-branching enzyme (I blood group)

NM_001491.2

-7.4

-2.5

-2.5

C14orf135

Pecanex-like 4 (Drosophila)

NM_022495.5

-7.5

-2.7

-2.5

PBK

PDZ binding kinase

NM_018492.2

-7.8

-3.3

-2.4

TOP2A

Topoisomerase (DNA) II alpha 170 kDa

NM_001067.2

-8.1

-3.8

-2.7

CAV2

Caveolin 2

NM_001233.3

-8.1

-2.3

-2.5

SERTAD2

SERTA domain containing 2

NM_014755.1

-8.3

-3.1

-3.6

ACSL4

Acyl-CoA synthetase long-chain family member 4

NM_004458.1

-8.5

-2.5

-2.5

FAM83D

Family with sequence similarity 83, member D

NM_030919.2

-9.2

-4.3

-3.8

CDK6

Cyclin-dependent kinase 6

NM_001259.5

-9.4

-3.1

-4.1

FRMD6

FERM domain containing 6

NM_152330.3

-9.5

-3.1

-2.4

SNAPC1

Small nuclear RNA activating complex, polypeptide 1, 43 kDa

NM_003082.2

-9.8

-2.3

-2.0

CALD1

Caldesmon 1

NM_033140.2

-9.9

-2.1

-2.5

BCAT1

Branched chain amino-acid transaminase 1, cytosolic

NM_005504.4

-10.2

-5.6

-4.2

DLGAP5

Discs, large (Drosophila) homolog-associated protein 5

NM_014750.3

-10.4

-3.1

-2.4

ANLN

Anillin, actin binding protein

NM_018685.2

-10.9

-3.1

-2.2

TACC1

Transforming, acidic coiled-coil containing protein 1

NM_006283.1

-11.1

-2.7

-2.6

AP1S2

Adaptor-related protein complex 1, sigma 2 subunit

NM_003916.3

-11.5

-2.5

-2.1

CTNNAL1

Catenin (cadherin-associated protein), alpha-like 1

NM_003798.2

-13.9

-3.1

-2.1

DCBLD2

Discoidin, CUB and LCCL domain containing 2

NM_080927.3

-14.5

-3.1

-2.6

CAV1

Caveolin 1, caveolae protein, 22 kDa

NM_001753.3

-20.1

-3.3

-2.8

We performed computational analysis on the 393 genes using String software to identify interaction networks that might help reveal functional nodes indicative of the biological response of these cells to fever range hyperthermia. Our analysis uncovered a remarkably dense interaction node centered on genes involved in mitotic progression (Figure 4). We performed Metacore analysis on the list of 393 genes, confirming that mitotic cell cycle regulatory networks exclusively dominated the top statistically significant pathway maps (Table 4 lists the top 20 identified networks). Figure 5 illustrates Metacore’s analysis of the interrelationships of the identified mitotic regulatory genes including STAG2, NEK2, KPNA4, IPO5, TNPO1, CCNB1, CDK1, CDK6, NCAPG, NCAPG2, TOP2A, NUF2, CENPE, CENPF, ZWILCH, PDS5A, WEE1, KIF11, CHUK, and PPP1CB. Of the 393 genes that were differentially expressed between the breast cancer and mammary epithelial cells following H’ to H analysis, approximately 80% of the top 60 most upregulated genes were histone clusters and non-protein coding RNAs such as small nucleolar-, ribosomal-, and micro-RNAs. These data cumulatively suggest that the selective disadvantage that breast cancer lines experience following mild hyperthermic shock may be due to an inability to correctly regulate their core biological processes and mitotic cell cycle machinery. The differential expression of genes involved in these processes for the H’ vs H analysis is shown in Figure 6.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-81/MediaObjects/12885_2013_Article_4319_Fig4_HTML.jpg
Figure 4

Interaction network analysis of the differential response of the breast cancer cells to fever range hyperthermia reveals a strong node centered on mitotic cell cycle progression. The list of 393 genes identified as differentially expressed in the breast cancer lines following fever range hyperthermia in the H’ vs H comparison were queried using String 9.05. Lines illustrate known physical and functional associations derived from previously reported genomic context, high-throughput experiments, coexpression analysis, and Pubmed.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-81/MediaObjects/12885_2013_Article_4319_Fig5_HTML.jpg
Figure 5

Interrelationship between the mitotic regulators that differentiate the hyperthermic response of breast cancer cells from mammary epithelial cells. Metacore analysis of the 393 genes that differentiate the hyperthermic response of breast cancer from mammary epithelial cells in the H’ vs H comparison identified mitotic cell cycle progression (and 20 associated mitotic regulatory genes) as the primary differential gene networks. We used Metacore to identify the interrelationship of the known physical and functional associations between these 20 genes (red markers).

https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-81/MediaObjects/12885_2013_Article_4319_Fig6_HTML.jpg
Figure 6

Gene expression changes in histones, non-protein coding RNAs, and mitotic regulators differentiate the hyperthermic response of breast cancer cells from mammary epithelial cells. Heatmap depicting the two-fold or greater changes in RNA expression levels (p < 0.05) for histone, non-protein coding RNA, and mitotic regulatory genes in the H’ vs H comparison (red?=?overexpressed, green?=?underexpressed).

Table 4

Top 20 significantly significant GeneGo pathway maps that are differentially expressed amongst all three breast cancer cell lines relative to the mammary epithelial line in the H’ vs H comparison

GeneGo Pathway

p-Value

Mitosis

8.15e-44

Cell division

3.02e-35

Cell cycle

5.37e-23

Mitotic sister chromatid segregation

1.31e-22

Mitotic spindle organization

4.87e-20

Protein localization to kinetochore

1.54e-19

Chromosome segregation

4.85e-16

Establishment of mitotic spindle orientation

7.53e-16

Mitotic cell cycle

9.66e-16

Homologous chromosome segregation

3.30e-13

Mitotic cell cycle checkpoint

5.99e-12

Anaphase promoting complex-dependent degradation

2.27e-10

Mitotic cell cycle spindle assembly checkpoint

2.03e-09

Spindle assembly involved in mitosis

4.44e-09

Mitotic anaphase

6.97e-09

Microtubule-based movement

3.57e-08

Spindle organization

4.83e-08

Mitotic centrosome separation

7.75e-08

Spindle assembly

1.92e-07

Altered expression of mitotic arrest genes differentiates the hyperthermic response of breast cancer cells from that of mammary epithelial cells

Our microarray analysis strongly suggests that the inability of breast cancer cells to regulate their mitotic cell cycle machinery may be a major contributing factor to their selective disadvantage following hyperthermia. Therefore we independently tested the expression levels of a panel of mitotic regulators that were identified as differentially expressed in the H’ vs H analysis. Quantitative real time PCR analysis of cDNA collected from the H’ vs H treatments for the steady state mRNA levels of several genes with core processes related to mitosis including KIF11, CDK6, STAG2, NEK2, CHUK, KPNA4, CENPF, and NCAPG correlated well with our microarray data, revealing differential expression of these genes for each cell line in the hyperthermia treatment relative to the normal temperature (Figure 7A). A comparison of the qPCR and microarray data for each of these selected genes for the H’ to H comparisons is depicted in Table 5. To confirm the hyperthermia-induced mitotic defect in the breast cancer lines, we subjected all four cell lines to 30 minutes of fever range hyperthermia (H and H’) or normal control temperature (C and C’) and collected the cells after 24 hours for cell cycle analysis using flow cytometry. The cells were collected 24 hrs after treatment as this is sufficient time to see the phenotypic effects on the cell cycle that would be induced by altered RNA expression. Propidium iodide staining of cells from each condition clearly revealed that a G2/M phase accumulation as a common event across all three breast cancer lines following hyperthermia even after 24 hours following the treatment, but did not occur in the mammary epithelial lines (Figure 7B). Collectively, these data provide evidence to suggest that the selective disadvantage of breast cancer cells in response to hyperthermia could be due, in part, to altered regulation of mitotic machinery following heat shock.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-81/MediaObjects/12885_2013_Article_4319_Fig7_HTML.jpg
Figure 7

Biological confirmation of gene expression analysis. (A) qPCR analysis measuring the fold change of mitotic regulators following hyperthermia treatment. RQ values are represented as the hyperthermia-induced change in gene expression for each gene relative to the expression of the same gene in the normal temperature condition (RQ?=?1). The data shown are the median of at least 3 replicates, plus or minus the standard deviation, and presented in log scale. (B) The panel of mammary epithelial and breast cancer cells were grown under standard growth conditions or treated with 30 minutes fever range hyperthermic shock. Cells were harvested after 24 hours and cell cycle analysis was performed using flow cytometric detection of propidium iodide intensity.

Table 5

qPCR and microarray expression data for selected cell cycle genes in the H’ vs H comparison

Gene Symbol

MCF7

MBA231

MBA468

 

qPCR

Array

qPCR

Array

qPCR

Array

KIF11

-1.5

-5.2

-2.6

-3.3

-1.2

-3.1

CDK6

-2.4

-9.4

-1.7

-3.1

-3.0

-4.1

STAG2

-2.7

-3.0

-3.2

-2.9

-2.0

-2.5

NEK2

-4.7

-4.6

-1.7

-2.7

-2.8

-2.6

CHUK

-4.3

-2.3

-1.4

-2.2

-1.3

-2.3

KPNA4

-1.7

-2.4

-7.3

-2.2

-1.6

-2.4

CENPF

-2.9

-7.4

-183

-2.9

-5.4

-2.7

NCAPG

-3.5

-4.2

-9.3

-2.7

-1.9

-2.2

p?≤?0.05 for all values via Student’s t-test.

Discussion

While hyperthermic treatment of tumors has been utilized since the time of the ancient Greeks and modern medicine has implemented hyperthermia as an adjuvant treatment in various settings, use of this technique has been marred with limitations including the inability to target heat to the tumor without collateral damage to the neighboring cells, homogenous heat dispersion throughout the entire tumor, and intrinsic problems with targeting undetectable micrometastases. In recent years, advances in nanoparticle-enabled thermal therapy hold the promise to overcome many of these issues, thus a strong interest in treatment of tumors with hyperthermia has been renewed. While it has been established for decades that normal tissues exhibit enhanced thermotolerance relative to cancer cells [1, 2], the mechanisms controlling this are largely unknown. Studies on the heat shock response of cancer cells have revealed changes in apoptosis, cell cycle regulation, and cell structure/maintenance [3], yet very little has been reported critically comparing the heat shock responses of cancer cells to their non-diseased cellular counterparts. Thus it is currently unknown at the molecular level how thermotolerance is maintained in normal cells, but lost or deregulated in cancer cells. To address this, we utilized a genomics approach to address two areas: 1) identify the global transcriptional response to hyperthermia of a panel of breast cancer and mammary epithelial cells using a C vs H and C’ vs H’ analysis and 2) compare the hyperthermia-induced changes in global gene expression patterns of the breast cancer cell lines to the mammary epithelial cells using a H’ vs H analysis. As a result of these studies, we identified several gene networks that reflect the hyperthermic response of breast cancer and mammary epithelial cells (including cell cycle, heat shock, survival/apoptosis, DNA damage and Rab/Ran regulation) and that clearly differentiate the response of breast cancer cells from that of mammary epithelial cells (including mitotic regulation and expression of histone and non-protein coding RNAs).

Evaluation of the hyperthermic response of breast cancer and mammary epithelial cells

C vs H and C’ vs H’ comparative analysis of the gene expression profiles of each cell line revealed that the mammary epithelial cells responded to increased temperature distinctly from the breast cancer lines, with altered regulation of gene networks controlling DNA damage response, cell cycle progression, apoptosis, and heat shock characterizing the mammary epithelial cell response. In contrast, the three breast cancer lines commonly altered gene networks encoding Rab and Ran G-protein regulators in response to hyperthermia.

Arguably the most studied response of cells to hyperthermia is that of heat shock protein activation and expression and one might guess that heatshock-protein mediated responses are likely responsible for the selective disadvantage of solid tumors to fever range hyperthermia. Numerous cell stresses have been shown to induce heat shock proteins, which act as molecular chaperones inside cells to modulate thermotolerance and protect cells from stress-induced death [2729]. MCF10A cells exhibited significantly increased expression of HSP90AA1, CARHSP1, HSPA12A and decreased expression of HSPB1, HSPBL2, HSPA6, and HSPA7 (C vs H), while the three breast cancer lines showed no significant 2-fold or greater alterations in the expression of these genes (C’ vs H’). Despite this finding, we provide evidence that suggests the ability of mammary epithelial cells to properly modulate their heat shock response does not contribute to the selective disadvantage of breast cancer cells to hyperthermia. For instance, comparison of heat shock protein expression in the H’ vs H analysis revealed no significant difference in the relative abundance of these heat shock protein genes regardless of the cell type. As elevated expression of heat shock proteins has been observed in various types of cancers [3032], hyperthermic shock may simply bring the heat shock protein expression in MCF10A cells to the baseline levels in the breast cancer lines.

Hyperthermia has long been known as an effective radio- and chemo-sensitizing agent and it would be an attractive hypothesis that hyperthermia may impart a selective disadvantage to breast cancer cells via upregulation of DNA damage or reduction in its repair. Indeed, hyperthermia has been shown to induce chromosomal damage during S-phase [33] and inhibit homologous recombination repair via a heat shock protein/Brca1/2 pathway [3436]. Furthermore, hyperthermia induces signaling pathways that overlap with those activated by ionizing radiation-induced DNA damage including histone H2Ax phosphorylation and enhanced ataxia-telangiectasia mutated protein (ATM) activity [37]. Analysis of C vs H and C’ vs H’ revealed a number of genes involved in DNA damage response whose expression was altered in the MCF10A cells, and similar changes were not observed in the three breast cancer lines. Despite this, no statistically significant changes in gene expression for these genes were observed in our H’ vs H comparison, suggesting that (similar to the heat shock proteins) this pathway may not clearly distinguish the selective disadvantage of breast cancer cells to hyperthermia.

Gene networks that distinguish the hyperthermic response of breast cancer cells from mammary epithelial cells

As our initial analysis compared only the heat shock response of each individual cell line relative to its transcriptional expression at normal growth temperature, we extended our analysis by directly comparing the transcriptional response of the H’ vs H treatments to identify the unique gene networks that clearly differentiate the gene expression changes unique to the breast cancer cells following heat treatment. This comparative analysis identified cell cycle networks preferentially involved in mitotic progression as well as large scale changes in the expression of histones and non-protein coding RNAs as the major distinctions between the hyperthermic responses between the breast cancer lines and the MCF10A cells. 80% of the top 60 genes commonly expressed at higher levels in the three breast cancer lines following heat shock relative to the mammary epithelial line following heat shock were histones and non-coding RNA. This effect was due primarily to decreased expression of these genes in the MCF10A cells with no change or only a small upregulation in expression in the breast cancer lines, suggesting that mammary epithelial cells are repressing many of their core processes (chromatin condensation, transcription, translation, etc.) following hyperthermic shock, while the breast cancer cells may continue performing these processes as normal. Similar findings have been reported following other cellular stresses whereby oxidative damage significantly decreases the expression of histones and ribosomal proteins [38]. In addition to histone gene expression, heat shock induces an array of chromatin post-translational modifications. For instance, HSP70 has been shown to enhance the phosphorylation of histone H3 following heat shock [39], and histone variant H3.3 has been shown to stimulate heat shock induced HSP70 transcription [40], suggesting that heat shock response and histone activity are tightly regulated.

Our data revealed that a number of small nucleolar RNAs, which play key roles in ribosomal biosynthesis, were differentially regulated between the mammary epithelial and breast cancer cells following hyperthermia. Several small nucleolar RNAs are reportedly critical mediators of oxidative stress and their overexpression has been associated with reduced resistance to oxidative stress [41, 42]. Small nucleolar RNAs have been shown to bind to the mature RNA of heat shock cognate protein (HSC70) [43] and inhibition of Hsp90 prevents the accumulation of U3 and U4 small nuclear ribonucleoproteins via a process that involves Pih1/Nop17 and R2Tp complexes [4446].

Changes in cell cycle progression (particularly mitotic catastrophe) have been repeatedly shown to characterize the hyperthermic response of numerous cell types [4749], though it is largely unstudied as to how cell cycle changes differ between normal and tumor cell lines following this treatment. Major distinctions in cell cycle networks involved in mitotic progression clearly distinguished the H’ vs H analysis of our data. The genes that were differentially expressed at statistically significant levels included those involved in spindle assembly and chromosome separation, chromosome condensation in prometaphase, metaphase checkpoint, sister chromatid cohesion, and initiation of mitosis. Our confirmatory experiments using flow cytometry further revealed that hyperthermia treated breast cancer cells stalled in the G2/M phase of the cell cycle within 24 hours post-treatment, while the cell cycle profiles of heat-shocked mammary epithelial cells were similar to those grown at normal temperatures. Interestingly, it has been shown that cells vary in their susceptibility to heat in accordance to their phase in the cell cycle, with the highest heat sensitivity observed during mitosis due to damage to the mitotic apparatus, leading to inefficient mitosis and polyploidy. M- and S-phase arrested cells show increased susceptibility to heat-induced damage, while G1-phase cells are relatively heat resistance [5053].

This study has been the first to shed light on the comparisons of transcriptome-level fever range hyperthermic responses of mammary epithelial cells to breast cancer cells. While this data points to a number of areas that potentially contribute to the selective advantage of normal breast epithelium over its malignant counterparts following hyperthermia, our studies were simplistic in that they utilized a cell culture monolayer system solely consisting of cells derived from normal or tumor breast tissue. We have gained solid insight into the responses of these particular cell types to fever range hyperthermia, however a tumor is a very complex entity. For instance, solid tumors are not only composed of the tumor cells, but also consist of endothelial, fibroblast, and immune cells which will each respond to hyperthermia in their own fashion and potentially affect the response of the tumor as a whole. Moreover, heterogenous heat distribution and dissipation due to a faulty tumor vascular system may induce uneven heating in the tumor itself, thus affecting some areas distinctly and differentially altering the tumor’s response to hyperthermia. Future studies should be undertaken to address these issues.

Conclusion

Collectively, our data suggest that fever range hyperthermia affects breast cancer cells distinctly from mammary epithelial cells. These differences are largely attributed to alterations in the expression of genes involved in mitotic cell cycle progression, histones, and non-coding RNAs. Considering the hyperthermia induced G2/M cell cycle defects observed in the breast cancer cells but not the mammary epithelial cells, these data pose the question as to whether hyperthermia may function in a synergistic manner when combined with drugs that specifically target mitosis such as taxols and vinca alkaloid derivatives.

Abbreviations

C: 

37°C treatment of mammary epithelial cells

C’: 

37°C of breast cancer cells

H: 

45°C of mammary epithelial cells

H’: 

45°C of breast cancer cells

RNA: 

Ribonucleic acid

DNA: 

Deoxyribonucleic acid

aRNA: 

Amplified RNA

cDNA: 

Complementary DNA

mRNA: 

Messenger RNA

G1: 

Gap 1

S: 

Synthesis

G2: 

Gap 2

M: 

Mitosis

RT PCR: 

Real time polymerase chain reaction.

Declarations

Acknowledgements

This research was supported by startup funding to BB from TTUHSC.

Authors’ Affiliations

(1)
Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center
(2)
Department of Physics, University of Texas

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  54. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/81/prepub

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© Amaya et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.