Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

Invasive ductal (IDC) and lobular carcinomas (ILC) are the most common histological types of breast cancer [1, 2]. The terminology of ductal and lobular carcinomas is considered to be controversial as on purely anatomical grounds there is no justification for this nomenclature. Both carcinomas are derived from the terminal duct lobular unit (TDLU), and the differences in their morphology are likely to reflect the differences in mechanisms of carcinogenesis rather than the anatomical origin of the lesions. At presentation the clinical pathological parameters such as tumor site, size, grade and stage are similar for both tumor types [3], however, clinical follow-up data and the patterns of metastasis suggest that their development and progression are different [4, 5]. Treatment for stage-matched tumors is similar [6], but ILCs are often resistant to neoadjuvant therapy [7]. Although patients with ILCs are older, have low grade tumor and less lymphatic invasion, they have no survival advantage compared with IDCs [8, 9].

Expression profiling using microarrays is a powerful technology which enables the simultaneous study of the expression of thousands of genes and, in conjunction with laser capture microdissection, the high-throughput genetic analysis of morphologically distinct cell subpopulations within tumor tissue [10, 11]. Microarray analysis has a number of applications, including tumor classification, molecular pathway modeling, functional genomics, and comparison of gene expression profiles between groups [12]. The study of gene expression in primary breast tumor tissues is complicated for two major reasons. First, breast cancer consists of many different cell types, including normal epithelial, stromal, adipose and endothelial cells. Second, tumor cells are morphologically and genetically diverse [13]. The recent development of laser capture microdissection has provided an opportunity to generate gene expression signatures from individual cell types [14–20].

Microarrays were used for analysis of breast tumor subclasses with clinical implication [21, 22], for analysis of gene expression changes in single breast cancer cells from within the same tumor [14], for expression analysis of different gene families in breast cancer [23, 24], and for analysis of gene expression in different cellular and tumor types [25–28]. ER status of the tumor was the most important discriminator of expression subtypes. Unsupervised hierarchical clustering segregated these tumors into two main clusters based on their basal (predominantly ER negative) and luminal (predominantly ER positive) characteristics [29]. Ductal breast cancer classes have been identified with aggressive phenotype and poor prognosis versus those with good prognosis [30–32]. Another study reported distinct expression patterns based on BRCA1 and BRCA2 status [33].

To date, few papers have been published on gene expression profiles of normal cell populations in the mammary gland [34]. Several studies suggest differences in expression profiles of IDC and ILC. Inactivating mutations of E-cadherin gene are very frequent in ILC [35]. However, the loss of E-cadherin expression was shown to be an independent prognostic marker for recurrence, especially in node-negative breast cancer patients, irrespective of the histological type [36]. Abnormal cytoplasmic and nuclear localization of p120, a member of the E-cadherin/catenin adhesion complex, is mediated by E-cadherin loss and occurs in the early stages of ILC [37]. Non-microdissected IDC and ILC tissues have also been used to study specific gene expression profiles of lobular and ductal tumors [38, 39]. We aimed to identify genes differentially expressed between normal cell types (ductal and lobular), between tumor cells and normal cells as well as between tumor types (ductal no special type and lobular). Despite examining limited number of patients, our study is the first full genome analysis of microdissected ductal and lobular tumor and normal cells which allowed us to detect normal mammary epithelium- and cancer-specific genes.

We examined 30 samples (microdissected tumor and normal ductal and lobular cells) from postmenopausal patients with lobular and ductal invasive breast carcinomas using Affymetrix arrays. Genes differentially expressed between the normal ductal and lobular cell types, which are less likely to be affected by fluctuating levels of female hormones as they were derived from postmenopausal women, are involved in ion transport and protein kinase cascade, protein biosynthesis and modification, proteolysis and metabolism, regulation of transcription and cell growth. However, hierarchical clustering of normal cell types based on the 82 probe sets identified by both RP and pairwise comparison showed that the gene expression profiles of normal ductal and lobular cells taken from the same patient are more similar to each other than ductal cells or lobular cells from different patients, and thus could not be well separated from each other. This may suggest expected similarities between expression signatures in normal epithelial (ductal and lobular) cells of the mammary gland. Since both IDC and ILC are believed to start in the terminal duct lobular unit (TDLU) of the breast [1, 2], and these normal cell populations showed similar gene expression profiles, it is likely that the different morphological appearances of the two tumor types are mediated by differences in their mechanisms of carcinogenesis.

Combined pairwise comparison and RP analysis revealed that ductal and lobular carcinomas have a number of genes in common, however, they can be discriminated both at the gene and protein levels in our study. cDNA microarrays have previously been used to distinguish between IDC and ILC [38, 39]. Unsupervised clustering of tumors failed to separate the two subtypes. There were 8 genes identified by MaxT permutation analysis using t tests, significance analysis for microarrays (SAM) and prediction analysis for microarrays (PAM) (E-cadherin, survivin, cathepsin B, TPI1, SPRY1, SCYA14, TFAP2B, and thrombospondin 4), and an additional 3 were identified by SAM and PAM (osteopontin, HLA-G, and CHC1) [38]. It has also been found that over half of ILCs differed from IDCs in global transcription programs, whereas the remaining ILCs closely resembled IDCs. Fifty two percent of the ILCs ("typical" ILCs) clustered together and displayed different gene expression profiles from the IDCs, whereas the other ILCs ("ductal-like" ILCs) were distributed between different IDC subtypes. Many of the differentially expressed genes encode for proteins involved in cell adhesion/motility, lipid/fatty acid transport and metabolism, immune/defense response, and electron transport. Many genes distinguishing between typical and ductal-like ILCs are involved in regulation of cell growth and immune response [39]. However, these two previous studies examined whole tumor tissues without microdissection, suggesting that expression of a number of genes could be related not only to tumor cells but also to other components of mammary tissue such as stromal, adipose, endothelial etc. Our study is the first full genome analysis of microdissected ductal and lobular tumor and normal cells reporting both normal mammary epithelium- and cancer-specific genes expression profiles.

Importantly, CDH1 (E-cadherin gene) was downregulated in our lobular carcinomas, and immunohistochemistry confirmed this loss at the protein level within tumors. E-cadherin is considered to be the most important cell adhesion molecule in the mammary gland. It acts as a tumor suppressor inhibiting invasion and metastasis. Mutations of this gene are correlated with gastric, breast, colorectal, thyroid and ovarian cancer. During tumor progression, E-cadherin can be functionally inactivated or silenced by different mechanisms such as post-translational control, somatic mutations, downregulation of gene expression through promoter hypermethylation, histone deacetylation, and transcriptional repression [52, 53]. The latter induces cellular responses leading to the conversion of epithelial cells into invasive mesenchymal-like cells with increased motility and invasiveness, and this process is called an epithelial-mesenchymal transition (EMT) [52]. To date, it is believed that lost, non-polar or cytoplasmic expression of E-cadherin protein and/or transcriptional repression of its mRNA are hallmarks of EMT in cancer progression [53–55]. It has also been shown that several proteins such as fibronectin and integrin αvβ6 [54], Ets, TGFβ, FGF-1,-2,-8, α-SMA, collagen type I, III and thrombospondins increase in abundance during EMT [56], conversely, amongst proteins that decrease in abundance are E-cadherin and cytokeratins [54]. According to our results, collagen type I and III, fibronectin and Ets domain transcription factor are upregulated and cytokeratins are downregulated in both tumor cell types. The expression of collagens and other mesenchyme-associated genes in microdissected breast cancer cells was also confirmed by Nishidate and co-workers [57]. In addition, pairwise comparisons revealed that thrombospondin 4 was upregulated only in lobular cancer cells, which agrees with the literature [38]. Thus we propose that the EMT plays a role in both tumor types but appears to be more important in lobular carcinomas. The EMT phenomenon seems to be promising because multiple molecules involved in EMT, such as receptor- and SRC-family tyrosine kinases, RAS and other small GTPases, can be envisioned as targets for anti-EMT therapy [58].

The tissue microenvironment, including the ECM-cell and cell-cell interactions, plays an important role in both normal mammary gland development and cancer. Neoplastic transformation of cells dramatically alters the synthesis of proteoglycans and other ECM proteins both in tumor and the surrounding matrix [59]. This can stimulate the growth and spread of tumor cells by decreasing the adhesive functions of the ECM [60]. Both tumor types examined show upregulated genes involved in tumor-ECM interactions, cell adhesion and migration processes including metastasis. Expression of majority of the proteins encoded by these genes is related to TGFβ or Wnt signaling, and both the TGFβ and Wnt pathways may affect ECM.

Asporin is a cartilage extracellular protein that has been reported to be associated with knee and hip osteoarthritis. This leucine-rich repeat protein was shown to interact with and inhibit TGFβ signaling which is thought to lead to insufficient quantities of aggrecan and type II collagen in osteoarthritis [61, 62]. Asporin was more upregulated in our lobular carcinomas when compared with ductal tumors as well as with normal cell types. Overexpression of asporin mRNA in lobular carcinomas was then confirmed by chromogenic in situ hybridization and PCR. In support of our findings, upregulation of this gene has also been described in microdissected androgen-independent prostate cancer cells using Affymetrix Human Genome U133A GeneChips [63]. The authors did not discuss it and importantly, asporin has not been related to carcinogenesis to date.

We have found another candidate gene, collagen triple helix repeat containing 1 (CTHRC1), which was upregulated in ILC in comparison with normal cells, and its expression was also validated by PCR. Aberrant expression of CTHRC1 has recently been reported in human solid tumors, including cancers of the gastrointestinal tract, lung, breast, thyroid, ovarian, cervix, liver, and the pancreas. It is associated with cancer tissue invasion and metastasis and potentially plays important functional roles in cancer progression, perhaps by increasing cancer cell migration [64]. TGFβ upregulates CTHRC1, versican, ADAM12, and downregulates SFRP1 and E-cadherin [65–67]. The loss of SFRP1 is known to be associated with breast cancer progression and poor prognosis in early stages [68], and a similar expression profile is seen in our study. SFRP1 is downregulated and versican is upregulated in both tumor types. Furthermore, pairwise comparison identified other overexpressed genes in lobular carcinoma such as SFRP2 and ADAM12. E-cadherin, which is downregulated in lobular cancer cells, can also be repressed by TGFβ-induced expression of transcription factor complexes [69]. TGFβ signaling inhibitors have been shown to prevent EMT, to inhibit mammary tumor viability and to block metastasis in various murine models [70]. According to these results, we propose that deregulated TGFβ signaling is likely to be more important in lobular carcinogenesis.

Wnt signaling molecules are also expressed in our samples in several comparisons. The Wnt proteins are small secreted glycoproteins which are involved in the self-renewal of stem cells and may be responsible for the maintenance of mature tissues [71]. On binding to Frizzled receptors, Wnts can activate canonical (β-catenin-dependent) and/or non-canonical (β-catenin-independent Wnt/planar cell polarity pathway and Wnt/Ca2+ pathway) Wnt signaling [72, 73]. Downregulation of frizzled related proteins has been described in breast cancer [68]. Of these, SFRP1 is downregulated in both types of our tumor cells, and genes involved in calcium regulation pathway are not significantly changed. The promoter of fibronectin 1 contains LEF/TCR-binding sites, making it a direct target of canonical Wnt signaling [59, 74]. Fibronectin 1 is also upregulated in both tumor types. However, there is a difference in Wnt signaling between ILC and IDC. SULF1 (sulfatase 1) is upregulated and MMP7 is downregulated only in IDCs. MMP7 is a confirmed Wnt target [75] and it has been shown to be activated in both canonical [76, 77] and non-canonical Wnt signaling [78]. Binding Wnt ligands to frizzled receptors is regulated by the 6-O sulfation-desulfation of cell surface heparan sulfates (HSs) by sulfatase 1. Sulfated HSs bind to Wnt ligand with high affinity and inhibit Wnt signaling. Sulfatase 1 removes 0–6 sulfates from HSs and reduces their binding to Wnt ligands which in turn allows the formation of functional Wnt-Frizzled complexes and thus promotes Wnt signaling [79, 80]. Frizzled-related proteins also have heparin-binding domains that promote the formation of Wnt-Frizzled complexes [81]. Although SULF1 is upregulated, SFRP1 and MMP7 are downregulated in ductal cancer cells, whereas SFRP2 and other Wnt molecules, such as DVL1 and WISP1, are upregulated in lobular cancer cells. This suggests that Wnt signaling is activated in ILC cells but not in IDC cells. Since mRNA level of β-catenin was not changed between normal and tumor cells, the expression of Wnt molecules appears to be β-catenin-independent, favoring the non-canonical Wnt signaling in ILC. There is evidence that Wnts acting through the non-canonical pathway can promote tumor progression [82, 83] which may also be true in ILC.

In addition to Wnt molecules such as DVL1, pairwise comparison revealed that EMP1, gene encoding a tumor-associated membrane protein involved in cell-cell interactions and proliferation control [84], was upregulated, whereas DDR1, epithelial-specific receptor kinase capable of binding Wnt5 and regulating the adhesion of mammary cells [85, 86], was downregulated in ILCs. Immunohistochemistry has also confirmed the same differential expression of these three proteins in IDC and ILC on tissue microarrays. Thus all the evidence suggests that the two tumor types can be distinguished both at the gene and protein levels. Specific changes in gene and protein expression are likely to reflect the differences in mechanisms of carcinogenesis as well as the specific histological and clinical characteristics of these tumors derived from the same anatomical compartment, TDLU.

Microdissection of normal and tumor cell types from the breast and full genome expression analysis by Affymetrix arrays allowed us to provide novel data on breast cancer. Invasive lobular and ductal breast carcinomas can be differentiated both at the gene and protein levels. Despite analyzing only thirty samples from ten patients, the results are in good accordance with previous literature [14, 38, 39, 57, 64]. Our data provide evidence for deregulated TGFβ and Wnt signaling accompanied by the overexpression of mesenchyme-associated genes like the collagens, asporin and others which might be occurring in conjunction with an altered EMT. We propose that deregulated TGFβ signaling and EMT phenomenon are involved in both tumor types, but they seem to be more important in lobular carcinomas which is in concordance with the loss of E-cadherin expression and their distinct morphology from ductal tumors. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1), which might be significant in mammary gland carcinogenesis and may also be important either in cancer diagnosis or therapy. Besides E-cadherin, the proteins validated on tissue microarrays by immunohistochemistry (EMP1, DVL1, DDR1) may represent novel tissue markers helpful in the differentiation of ductal and lobular cancers. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis, and the optimum treatment strategies.