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Activation of SNAT1/SLC38A1 in human breast cancer: correlation with p-Akt overexpression
- Kuo Wang†1,
- Fang Cao†1,
- Wenzheng Fang†2,
- Yongwei Hu1,
- Ying Chen3,
- Houzhong Ding1Email author and
- Guanzhen Yu2Email author
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 30 November 2012
Accepted: 14 May 2013
Published: 12 July 2013
SNAT1 is a subtype of the amino acid transport system A that has been implicated to play a potential role in cancer development and progression, yet its role in breast cancer remains unclear. In present study, we detected SNAT1 expression in breast cancers and explored its underlying mechanism in promoting breast carcinogenesis.
RT-PCR and Western blotting were performed to analyze the transcription and protein levels of SNAT1 in breast cancer cell lines and fresh tissues. Tissue microarray blocks containing breast cancer specimens obtained from 210 patients were constructed. Expression of SNAT1 in these specimens was analyzed using immunohistochemical studies. SNAT1 was down-regulated by SNAT1-shRNA in breast cancer cells and the functional significance was measured.
SNAT1 was up-regulated in breast cancer cell lines and breast cancer tissues. Overexpression of SNAT1 was observed in 127 cases (60.5%). Expression of SNAT1 was significantly associated with tumor size, nodal metastasis, advanced disease stage, Ki-67, and ER status. Suppression of endogenous SNAT1 leads to cell growth inhibition, cell cycle arrest, and apoptosis of 4T1 cells and lowered the phosphorylation level of Akt. SNAT1 expression correlated significantly with p-Akt expression in human breast cancer samples.
The cross-talk between Akt signaling and SNAT1 might play a critical role in the development and progression of breast cancer, providing an important molecular basis for novel diagnostic markers and new attractive targets in the treatment of breast cancer patients.
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death among females worldwide . Due to early detection, progress in treatment strategies and advances in our understanding of the molecular mechanisms of breast cancer, therapeutic effect increases and patients have longer survival duration. Unfortunately, global breast cancer incidence is increasing and most of these patients inevitably die of cancer recurrence and metastasis . Therefore, it’s essential to unveil the underlying mechanism of tumor progression and develop effective therapeutic strategies. So far, several oncogenic kinase signaling pathways have been considered as potential targets for cancer treatment. Among these pathways, PI3K/Akt/mTOR signaling has been shown to regulate cell proliferation, growth, migration and energy metabolism [3–5]. Activation of Akt and its clinical value have been widely reported in human breast cancer [4–7]. Recently, researchers show that the amino acid carrier plays an important role in various cell life activities, including energy metabolism, detoxication,neutrotransmission and most importantly malignant transformation of mammal cell. L-type amino acid transporter 1 (LAT1), for example, was widely investigated in various human solid tumors and increased expression of LAT1 was shown to be associated with tumor size, advanced disease stages, and Ki-67 labeling index and consequently with poor patient outcome [8–10]. Given the importance of Akt pathway and amino acid transporters in nutrients and energy metabolism of tumor cells, we hypothesized that Akt activation might be associated with up-regulation of amino acid transporters .
Among these amino acid transporters, system A has been found to be overexpressed in human solid cancers, including glioma , hepatoceller carcinoma  and hilar cholangiocarcinoma . System A amino acid transporter has three members: SNAT1, SNAT2, and SNAT4 (previously referred to as ATA1, ATA2, and ATA3, respectively), encoded by the SLC38 gene family (Slc38a1, Slc38a2, and Slc38a4) [15–17]. Among these three members, SNAT1 was significantly elevated in hepatocellular carcinoma and cholangiocarcinoma [13, 14]. Knocking down endogenous SNAT1 inhibited cell proliferation of HepG2 cells . Moreover, SNAT1 expression significantly correlated with tumor recurrence and poor outcome of patients with changiocarcinoma . However, the expression pattern of SNAT1 and its role in breast cancer development has not been fully demonstrated.
In the present study, we sought to determine the expression profiles of SNAT1 in breast cancers and cells and to investigate its correlation with p-Akt. In vitro, we further confirmed the association between SNAT1 expression and Akt activation, which controlled cell viability and colony formation.
Recombinant murine EGF was purchased from PeproTech Inc. (Rocky Hill, NJ). phospho-Akt (Ser473) antibody was purchased from Cell Signaling Technology (Beverly, MA). Anti-SLC38A1 antibody was from Abcam Company (Cambridge, UK). ß-actin and Ki-67 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell lines and culture conditions
The breast cancer cell lines MCF-7, MDA-MB-231 and 4T1 were purchased from the Cell Center of Chinese Academy of Sciences, Shanghai, China. MCF-7, MDA-MB-231 and 4T1 were maintained in DMEM with 10% fetal bovine serum (FBS) (Invitrogen Corp., Grand Island, NY). The cell lines were cultured in a 37°C humidified atmosphere containing 95% air and 5% CO2.
Tissue samples and tissue microarray construction
Association between SNAT1 and p-Akt expression and clinicopathologic factors in breast cancer
Five paraffin-embedded tissue microarray blocks of normal and tumor tissue specimens obtained from these patients were created using a manual arrayer (Beecher Instruments, Sun Prairie, WI, USA). Forty-five cases had one 1.5-mm core of nonneoplastic tissue and two 1.5 mm cores of primary tumor tissues. The other cases only had two 1.5 mm cores of primary tumor tissue. Besides, four fresh breast cancer tissues and matched fresh nonneoplastic tissues were used to detect the expression levels of SNAT1 mRNA and protein. Ethical review committees (Institutional Review Board of the Affiliated Kunshan First People’s Hospital, Jiangsu University and Institutional Review Board of Changzheng Hospital, Shanghai) approved the use of all tissues and clinical information (KS2008-01 and CZEC2001-01).
RNA preparation and reverse transcription-polymerase chain reaction
Total RNA was isolated from breast cancer cell lines and homogenised breast cancer samples using the AB gene Total RNA Isolation Reagent (Advanced Biotechnologies Ltd., Epsom, Surrey, UK). RNA concentration and quality were determined by spectrophotometric measurement (WPA UV 1101, Biotech Photometer, Cambridge, UK). cDNA was generated from 1 ug of each RNA sample and a reverse transcribed using a transcription kit (Takara, Kyoto, Japan). mRNA levels of SNAT1were assessed using the specific oligonucleotide primer pairs SNAT1 (sense: CCAGTGGCCTAGCTGGTACCAC and antisense: TCCCCAGCGAAAGTTGACTCAGAC); As an internal control, we used the β-actin primers (sense: GCTGTCACCTTCACCGTTC and antisense: CCATCGTCCACCGCAAAT).
Immunohistochemical analysis and evaluation of immunostaining
4 μm sections of paraffin-embedded tissue microarrays blocks were prepared and processed for SNAT1 (dilution 1:50, ab59721; Abcam, Cambridge, UK) and p-Akt (dilution 1:50, 736E11; CST, Beverly, MA) proteins staining. A S-p kit (KIT-9710; MAIXIN, Fuzhou, China) was used to visualize antibody binding on the slides. Counterstaining was performed with hematoxylin. All slices were evaluated without knowledge of the expression of another marker. SNAT1 and p-Akt protein expression in the 210 cases was evaluated by two individuals (C.Y. and G.Y.) under an Olympus CX31 microscope (Olympus, Center Valley, PA).
The expression of the proteins was evaluated using a semiquantitative scoring system. Staining was graded on a scale of 0–2 (0=negative staining [no staining of any tumor cells], 1=weakly expression [staining of <25% of tumor cells], and 2=high expression [staining of ≥25% of tumor cells]). Only a score of 2 was regarded as overexpression . Staining was scored independently by two individuals who were blinded to each other’s findings.
Western blot analysis
Breast cancer cell lines, breast cancer specimens and matched non-tumor tissues were prepared for Western blot analyses. Standard Western blotting was performed using a rabbit antibody against human SNAT1 (1:1000) and p-Akt (1:1000) and an anti-rabbit IgG antibody, which was a horseradish peroxidaselinked F(ab’)2 fragments obtained from a donkey (Amersham). Equal protein sample loading was monitored by probing the same membrane filter with an anti-β-actin antibody.
Plasmids and transfections
The shRNA-SNAT1 and unspecific scrambled shRNA plasmids were purchased from Genechem Company, Shanghai, China. At 24 hours before transfection, 1×105 cells were seeded in six well plates. Transfection of shRNA was carried out using Lipofectamine™ 2000 reagent (Invitrogen, Karlsruhe, Germany) and 4 ng shRNA plasmid per well according to the manufacturer’s instructions.
Cell proliferation assay
At 12 hours after transfection, cells were digested and 5000 cells were seeded in 96-well plates and incubated in medium with 10% FBS. At 24 h, 48 h, and 72 h, CCK8 assay (Dojindo Kumamoto, Japan) was performed to measure the final results. The experiment was repeated three times independently.
Colony formation assay
At 24 hours after transfection, cells were digested and seeded in 6-well plates in triplicate at a density of 500 cells/well for 14 days at 37°C. The colonies were fixed with methanol/acetone (1:1) and stained with crystal violet. Colonies with cell numbers of more than 50 cells per colony were counted.
Flow cytometric analysis
Flow cytometric analysis was performed as described previously to determine the effects of SNAT1-shRNA on cell cycle distribution and apoptosis [19, 20]. Briefly, 4T1 cells, grown in 6-well plates (2 × 105 cells/well), were synchronized at the G1/S boundary after starvation with basal medium for 24 hours, followed by transfection with SNAT-shRNA or shRNA vector for 48 hours. At the indicated time, cells were harvested by trypsinization and fixed with 70% ethanol, and measured following the manufacturer’s protocol (KEY GEN, Nanjing, China). Cell cycle distribution and apoptosis was analyzed by flow cytometry (FACSCalibur, BD Biosciences, Bedford, MA).
Statistical analysis was performed using the SPSS 16.0 statistical software program for Microsoft Windows. Categorical data were analyzed using χ2 statistics tests. Within-group correlations of continuous and ordinal variables were assessed using Pearson’s R correlation coefficient or Spearman correlation coefficient when appropriate. The Kaplan-Meier method was used to estimate survival rates, and the log-rank test was used to assess survival differences between groups. The significance of the in vitro results was determined by using the Student t test (two tailed). Two-sided P value <0.05 was considered statistically significant.
SNAT1 expression in patients with breast cancer
Correlation between SNAT1 expression and clinicopathologic characteristics of breast cancer
According to SNAT1 expression, the breast cancer patients were divided into two groups: SNAT1 negative expressers (n=83) and SNAT1 positive expressers (n=127). Table 1 summarized the correlation between SNAT1 overexpression and clinicopathological parameters in breast cancer. No significant relationship was found between SNAT1 expression and age, HER2, and PR expression. However, a statistically significant association was observed between SNAT1 expression and tumor size, lymph node metastasis, disease stage, Ki-67, and ER. Activation of SNAT1 occurred more frequently in large breast tumors (invasion to level T3-T4) (94.4%) than in small ones (level T1-T2) (53.4%; P<0.001) and more frequently in breast cancer with regional LN metastasis (93.2%) than in N0-stage tumors (42.6%; P<0.001). In regard to TNM stage, overexpression of SNAT1 was significantly associated with advanced disease stage: 97.6% at stage III /IV and 50.6% at stage I/II (P<0.001). Moreover, SNAT1 upregulation correlated significantly with Ki-67 overexpression (P=0.003) (Additional file 1: Figure S1) and ER-negative expression (P=0.002).
Knockdown of SNAT1 by shRNA induces cell growth inhibition and apoptosis of breast cancer cells by blocking Akt phosphorylation
Co-expression of p-Akt and SNAT1 in breast cancer specimens
Correlation between SNAT1 and p-Akt expression in breast cancer
Overexpression of SNAT1 and p-Akt on survival in patients with breast cancer
In this study, we found that up-regulation of SNAT1 was significantly associated with (1) tumor size, lymph node metastasis and advanced disease stage, the most important clinical determinants of treatment and prognosis for breast cancer; (2) Ki-67 overexpression and negative ER expression, the most important biomarker guiding treatment and outcome for breast cancer; (3) elevated activity of Akt, determined by the expression of phosphorylated Akt (p-Akt). Moreover, knockdown of SNAT1 blocked phosphorylation of Akt and hence attenuated cell growth and induced apoptosis of human breast cancer 4T1 cells. Therefore, we provided novel molecular evidence that activation of SNAT1/Akt signaling may play a critical role in breast cancer development and progression.
Glutamine has various important functions in mammalian cells and glutamine transport across cell membranes has been extensively studied physiologically . The glutamine transporter (ATA1/SNAT1/SAT1/SLC38A1) is a member of the system A transporter superfamily, providing metabolic fuel or precursors for glutathione synthesis . Physiologically, this carrier is mainly distributed in placenta and brain tissues [21, 23]. Recently, researchers revealed overexpression of SNAT1 in human solid malignant tumors, including hepatic carcinoma and changiocarcinoma [13, 14]. In the present study, SNAT1 expression was increased in breast cancer cells and tumor specimens compared with normal tissues at both mRNA and protein levels, suggesting oncogenetic role of SNAT1 in breast carcinogenesis. This result was further confirmed by immunostaining, which revealed a higher expression of SNAT1 in 60.5% cancer specimens and a lower expression of SNAT1 in 11.1% paraneoplastic tissues. Meanwhile, SNAT1 overexpression is closely correlated to tumor size, lymph node metastasis, disease stage, Ki-67, and ER-negative expression, indicating that SNAT1 is particularly important to breast cancer progression. Interestingly, patients with SNAT1 overexpression had a poor outcome than those without SNAT1 overexpression, further supporting the potential role of SNAT1 in cancer development and suggesting SNAT1 as a good target for cancer therapy. However, the relatively short follow-up duration limited the exploration of SNAT1 as an independent predictor of survival of breast cancer.
We then discovered that the knockdown of SNAT1 by specific shRNA reduced the viability of 4T1 cells. This inhibition might due to cell cycle arrested and apoptosis induced by downregulating SNAT1. Our data are in line with a study showing that siRNA mediated suppression of endogenous ATA1 lowered the viability of HepG2 cells . Take together, these siRNA or shRNA experiments suggested the SNAT1 molecule is essential in maintaining tumor survival. It has been shown that maternal protein restriction in rat inhibits Akt/mTOR signaling and down-regulates SNAT1 protein expression . In this study, p-Akt level was downregulated after transfection with SNAT1-shRNA in 4T1 cells. In particular, this inhibition was also observed for EGF-induced increase of p-Akt. These results provided a molecular basis for cross-talk between Akt and SNAT1.
Activation of AKT in human cancers induces multiple downstream cascades to promote cell survival, tumor growth and progression. Deregulation of AKT signaling was widely found in variety of human cancers including breast cancer. Previous studies demonstrated that overexpression of p-Akt was found in 30%~80% of cases with breast cancers [4–7]. Similarly, our data showed that p-Akt overexpression was observed in 64.3% cases and correlated significantly with tumor size, lymph node metastasis, disease stage, and ER-negative expression. Interestingly, we found that p-Akt expression co-localized with SNAT1 expression in caner specimens from the same patients. SNAT1(+)/p-Akt(+) was predominantly found in 120 cases, while SNAT1(−)/p-Akt(−) was in 68 cases, accounting 89.5% of all cases. This notion is supported by a recent study showing that Akt/mTOR signaling pathways and amino acid transporter activity can be simultaneously down-regulated by chronic maternal infusion of full-length adiponectin in pregnant mice . Other studies also revealed an interaction between other amino acid transporters (SLC36A1 and LAT1) and Akt signaling pathway [26, 27]. Taken together, the cross-talk between Akt and SNAT1 might play a critical role in cell growth and tumor metastasis. However, whether decreased expression of p-Akt is a feedback of cell growth inhibition by knocking down SNAT1 or a direct downstream target of SNAT1 needs further investigation.
In summary, SNAT1 was frequently activated in human breast cancer and its overactivation/overexpression was associated with advanced tumor stage and nodal metastasis. Additional in vitro study revealed that knockdown of SNAT1 inhibited cell growth inhibition, cell cycle arrested, and apoptosis of 4T1 cells by blocking the phosphorylation of Akt. The cross-talk between Akt signaling and SNAT1 provides an important molecular basis for novel diagnostic markers and new attractive targets in the treatment of breast cancer patients.
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