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Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy number gains. Through fluorescence in situ hybridization, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21-69%, median 55.5%, n=19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70-100%, median 93%, n=41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a post-treatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. Additionally, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and post-treatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment.

Background: Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling. Methods: In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo. Findings: CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ). Interpretation: CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling. Funding: Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research. Keywords: Breast cancer metastasis, Gamma subunits, L-type channels, Voltage-gated calcium channels (VGCCs)

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development., Studying the spatial mutational and gene expression alterations in breast cancer could impact our understanding of breast cancer development. Here, the authors analyse a unique dataset of epithelial samples that highlight potential field cancerisation surrounding the primary tumour.

Introduction Identification of driver mutations at single gene level and determination of their respective contribution to the enhanced invasive potential of cancer cells, via expression profiling and functional assays, are indispensable steps toward elucidating breast cancer pathobiology. In our previous array comparative genomic hybridization studies, gain in a region of chromosome 17, 17q23.3-24.3, was frequently observed in invasive duct carcinoma (IDC) patients with lymph node metastasis. Particularly, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1), found in 17q24.2, was associated with higher histological grade, larger tumor size, positive HER2 staining, and poorer prognosis from the analysis of a NKI dataset. PITPNC1 is involved in signal transduction and intracellular lipid transport, and may be an essential part of the epidermal growth factor signaling pathway. Additionally, amplification of PITPNC1 via the loss of microRNA-126 suppression has been implicated in metastatic angiogenesis and colonization. In this study, we aim to assess the role of PITPNC1 in the development of aggressive breast cancer by evaluating its effects on breast cancer progression and invasion. Method A total of 21 samples - 13 pure duct carcinoma in situ (DCIS), 8 IDC with or without lymph node metastasis - were used for the preliminary round of this study. Formalin-fixed and paraffin-embedded tissue blocks were microdissected and whole genome amplified using ligation-mediated PCR. Amplified DNA was subjected to quantitative real-time PCR. Copy number alterations of PITPNC1 were calculated with Livak method, using B2M as the reference gene. Lipofectamine transfection overexpressing PITPNC1 in non-invasive MCF-10A and invasive MCF7, MD-MB-231 cell lines was prepared for subsequent in vitro assays. Proliferation and migration capabilities associated with PITPNC1 overexpression is being evaluated using MTT assay and transwell migration assay. Result From preliminary qPCR data, PITPNC1 was shown to be amplified in DCIS samples (p = 0.0025) and IDC samples (p = 0.0093). The 95% confidence intervals of fold difference against normal breast tissue control are [7.1, 23.4] and [5.7, 24.4] respectively. PITPNC1 overexpressing MCF-10A cells exhibit a higher proliferation rate in culture compared to controls. Conclusion Consistent amplification of PITPNC1 is seen in both DCIS and IDC cases. However, it is still unclear if PITPNC1 amplification is a result of a single mutational event occurring early in cancer progression or a cumulative effect occurring over time. Additional qPCR on paired samples against a pooled control would be necessary to determine its contribution to invasion. The regularity of copy number gain in the PITPNC1 locus and its association with HER2 expression highlight its predictive potential as a novel biomarker for tumorigenesis or invasion. Citation Format: Peiqi Wang, Ranju Nair, Nisha Kanwar, Grace Cheung, Susan J. Done. Amplification of PITPNC1 affects breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1534.

Introduction: Several molecular markers have been investigated as possible prognostic factors for ductal carcinoma in situ (DCIS) progression to invasive ductal cancer (IDC). DCIS is regarded as a non-obligate precursor of IDC as it is frequently found adjacent to or surrounded by invasive breast cancer. Previously, we carried out genomic analyses that demonstrated that DCIS has specific genetic alterations that were conserved in invasive disease. Nevertheless, it is still unknown whether DCIS is merely a marker of increased risk, or actually a driver of the progression to IDC.Thus, it is crucial to better understand the genetic aberrations that underlie DCIS progression to IDC. Methodology: We conducted array chromosomal genomic hybridization on pure DCIS, IDC and DCIS paired with IDC, with 30 cases each (all were microdissected). Three chromosomal regions were selected based on the rate of their detection, which was significantly different (p Results: 61 samples were analyzed by comparing DCIS and IDC copy numbers of genes within the selected chromosomal regions. The median relative gene quantities were 1.66(4p15.33), 1.34(5q11.2) and 1.25(5q31.3). In all regions, IDC showed higher levels of amplification than DCIS with varying degrees of significance. Not all differences between DCIS and IDC in individual genes were significant. This observation may be attributed to focal variations in gene copy numbers and noise from Q-RT-PCR methods. 4p15.33 and 5q31.3 regions demonstrated significant gain in IDC compared to DCIS (p = 0.021). Genes within these regions include miRNA 572 (MIR572) and integrin alpha 2 (ITGA2). MIR572 is known to be involved in post-transcriptional regulation of gene expression, whereas ITGA2 encodes the alpha subunit of a transmembrane receptor for collagens. In the Chromosome 5q31.3 region, paired samples of DCIS and IDC also showed a trend in gene copy numbers, with IDC having a higher value than DCIS. Conclusions: Our findings demonstrate that there are specific genetic alterations associated with DCIS progression to IDC. In particular, MIR572 within Chromosome 4p15.33 and ITGA2 within Chromosome 5q31.3 may serve as candidate regions to predict the progression of DCIS to IDC. Note: This abstract was not presented at the meeting. Citation Format: Trillium E. Chang, Keisha Warren, Ranju Nair, Tian Y. Lu, Adewunmi Adeoye, Vladimir Iakovlev, Susan J. Done. Genomic alterations in ductal carcinoma in situ compared with Invasive breast cancer: a quantitative real-time PCR study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2015-4747

Introduction: We have previously found genomic gain on 17q22-24.2 to be a predictor of invasion when detected in duct carcinoma in situ (DCIS), and of nodal metastasis when detected in invasive duct carcinoma (IDC). Within this gene-rich amplicon we have identified a gene, Musashi homolog 2 (MSI2), which appears to play an important role in breast cancer progression. MSI2 is known to have a functional role in neural stem cell maintenance and the regulation of hematopoietic stem cells. More recently, this gene was shown to facilitate progression from the leukemic blast phase to the crisis phase. Methodology: The mRNA expression profile for MSI2 was determined by qRT-PCR in 9 breast cancer cell lines (MDA-MB-157, MDA-MB-231, MDA-MB-468, BT549, CAMA1, Hs578T, MCF7 and SKBR3) and a non-malignant mammary epithelial cell line (MCF10A). Western blots were used to confirm protein expression. MCF7 mammospheres and MSI2 overexpression clones were assessed for breast stem cell markers. Immunofluorescence was conducted at embryonic stages E10.5 to E15.5 to determine the role of MSI2 in murine mammary bud formation. MDA-MB-231 and MCF7 cells expressing MSI2-GFP and GFP clones were plated on transwells to examine cell migration and invasiveness. Proliferation was analyzed by the MTS assay. To understand the expression of MSI2 in patient tissue, tissue microarrays (TMAs) containing 232 breast cancers were analyzed by immunohistochemistry. Results: MSI2 was expressed in all breast cell lines tested. Immunohistochemical analyses of MSI2 in normal adult human breast tissue revealed expression in the basal luminal epithelial cells of the terminal duct lobular unit, with little expression in the stroma. In murine mammary line formation, E10.5 to E12.5 showed expression of MSI2 in both the mammary ductal epithelium and the mesenchyme. MSI2 expression diminished in mesenchyme and was restricted to the ductal epithelium by E14.5. MDA-MB-231 and MCF7 cells expressing MSI2-GFP demonstrated a 50% increase in cell migration and 30% more invasion compared to the GFP control. Correspondingly, knockdown clones showed the reverse effect. Increased MSI2 was coupled with an increase in the stem cell markers: CD24, CD44, Aldh1α1, CD133 and ABCG2 receptor. When the study was extended to patient TMAs, high MSI2 expression correlated with higher tumor grade. Conclusion: These experiments provide strong evidence that MSI2 plays an important role in normal breast development and breast cancer. MSI2 promotes migration and invasion and also appears to increase stem cell related properties which may explain the relationship to higher tumor grade. MSI2 is likely one of the drivers of the 17q22-24.2 amplicon in breast cancer and a better understanding of its role in breast cancer may lead the way to novel therapeutic strategies. Citation Format: Moustafa Abdalla, Ranju Nair, Nisha Kanwar, Nicholas Holzapfel, Juan C. Moreno, Susan J. Done. Characterization of the MSI2 gene on 17q22-24.2 and its role in Breast Cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1814. doi:10.1158/1538-7445.AM2013-1814

Introduction: Previously, we used aCGH to analyze genomic alterations in primary breast tumors with and without lymph node (LN) metastasis. A significant correlation was found between gain of chromosome 17q24 and LN metastasis. Selected genes including L-type calcium channel voltage dependent gamma subunit 4 (CACNG4) were further validated for increased protein expression in invasive breast tumors. CACNG4 is involved in calcium channel regulation in excitable cells of muscles and the brain. It is the only gamma subunit that also shows expression in non-excitable cells, such as glial and intestinal epithelial cells, suggestive of additional functions. It is over-expressed in glioblastoma multiforme cell lines resistant to EGFR inhibitors. Breast cancer gene expression datasets (NKI and IPC) identified an association of CACNG4 expression with LN metastasis and ER positivity. In the present study, we characterized CACNG4 in breast cancer with particular attention to its metastasis-related ability. Methodology: RNA interference was used for knockdown studies in cell lines followed by cell migration, invasion, aggregation and adhesion assays. Intracellular calcium influx in response to calcium channel agonists and antagonists was measured using the Fluo-4 calcium indicator dye. Results: CACNG4 showed variable expression across a panel of breast cancer cell lines. MCF10A (a non malignant cell line) does not express CACNG4, while it is highly expressed in ER positive cell lines (MCF7, MDA-MB-361, CAMA1) and lower in EGFR high cell lines (MDA-MB-468 and MDA-MB-231). CACNG4 knockdown inhibited cell migration and invasion in MCF7 and MDA-MB-231 cells. Conversely, cell adhesion and cell-cell aggregation was increased. RT-PCR revealed an inverse relation between CACNG4 expression and PKC-zeta specifically, which is involved in tight junction formation and shows low expression in breast, prostate and renal cancers. Thus, CACNG4 may play a role in de-adhesion and cell dissemination during metastasis. Blocking calcium channels with amlopidine had an anti-proliferative affect. Serum stimulation of EGFR high cell lines resulted in calcium influx, that was blocked in a dosage dependent manner with amlodipine, and synergistically blocked further with the EGFR specific tyrosine kinase inhibitor tryphostin AG1478. Knockdown of CACNG4 resulted in increased calcium influx suggesting a role in keeping the channel closed. Cells also showed resistance to amlodipine, requiring higher doses to block calcium influx. Conclusions: L-type channels are active in non-excitable breast cancer cells, and are affected by EGFR activity. CACNG4 maintains these channels in a closed state, modulating calcium influx, and inhibiting PKC-zeta expression which may result in metastatic function. The ability of calcium channel blockers to target channels in a CACNG4 dependent manner suggests novel treatment possibilities for invasive breast cancer. Citation Format: Nisha Kanwar, Ranju Nair, Dong-Yu Wang, Susan J. Done. The calcium channel subunit CACNG4 plays a role in breast cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5116. doi:10.1158/1538-7445.AM2013-5116

Metastasis of cancerous cells to distant sites is the major cause of death from breast cancer. In a previous study, we performed aCGH on breast invasive duct carcinoma samples with and without lymph node metastases and identified regions of genomic amplification that are associated with lymph node metastasis. These were subsequently validated by q-PCR. Within the 17q22-24 region, we have identified four genes likely to contribute to metastasis based on published data: COIL, WIPI1, TMEM100, and SLC16A6. We are using RNAi and ectopic over-expression to characterize these genes with in vitro functional assays for proliferation, migration, invasion, and survival. We used qRT-PCR analysis on a panel of seven breast cancer cell lines of varying invasiveness: MDA-MB-157, MDA-MB-231, MDA-MB-468, BT-549, CAMA-1, HS578T, MCF-7, and SKBR-3. We identified that WIPI1 and COIL were expressed in the majority. WIPI1 is up-regulated in a variety of tumor cells; studies suggest WIPI1 can activate autophagy by suppressing TORC1. Autophagy can inhibit cancer by maintaining genome stability via disposing of damaged mitochondria and peroxisomes. In clinical data obtained from 298 breast cancer patients, we observed high expression of WIPI1 to be associated with higher survival and lower breast cancer relapse. Alternatively, autophagy can also provide a survival mechanism for tumors with restricted blood supply and resistance to chemotherapy. This is in agreement with our observation of high WIPI1 protein expression in highly invasive breast cancer cell lines. We are over-expressing WIPI1 in SKBR-3 and knocking it down in BT-549 in vitro. WIPI1 could help determine the role of autophagy in tumor initiation processes such as proliferation and invasion, and in maintenance of tumor cells following therapy. COIL is found in a diffuse nucleoplasmic pool and in Cajal bodies (CBs). The role of diffuse COIL is still unclear, but CBs, involved in the formation of macromolecular complexes and found more in cells with high transcriptional activity, require COIL for proper formation and localization. We observed high expression of COIL protein in cell lines with higher proliferation index, noticeably in ER positive cell lines such as CAMA1 and absence in a non-tumorogenic cell line MCF10A. We are investigating the association of COIL with proliferation, invasion and migration abilities by over-expression and knockdown of the COIL protein in MDA231 and MCF7 breast cancer cells. We found from clinical data obtained from 245 breast cancer patients that high expression of COIL resulted in lower survival and a higher risk of relapse. This suggests that COIL may be a potential therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 93. doi:1538-7445.AM2012-93