Biological and therapeutic aspects of breast cancer progression

Abstract: Breast cancer is the second leading cause of cancer related death in women worldwide1. Although majority of primary breast cancers are curable with current treatment strategies, treatment outcome of metastatic breast cancer is dismal. The main focus of my doctoral studies is to investigate the causes of breast cancer recurrences and to eventually improve the survival outcome of metastatic breast cancer. Several factors has been attributed for the recurrence of breast cancer such as presence of cancer stem cells (CSCs) and the ongoing genomic evolution of cancer cells leading to intra tumor heterogeneity, which can in turn give rise to therapy resistant subclones. In this thesis we sought to investigate these two factors using breast cancer specimens. Firstly, in paper I, we optimized the method called “superficial scraping from tumor”. Using this method we were able to isolate epithelial breast cancer cells from which we can generate CSCs with ultra-low attachment and serum free conditions. Mammospheres generated from scraping material phenotypically resemble CSCs with ALDH1+, CD44+, and CD24- expression. Apart from CSC generation, scraping method can be used to biobank small tumors for future research purposes, without compromising routine histopathological analysis of patient samples. Next, we evaluated the expression of second estrogen receptor ERβ and its role in patient derived CSCs (Paper II), using the method optimized from paper I. We found that ERβ was predominantly expressed in both normal mammary stem cells (MSC) and CSCs. ERβ was found to be crucial for cancer stem cell phenotype and stimulation of ERβ using specific agonist increased mammosphere formation. Microarray analysis on ERβ stimulated MCF7 derived mammospheres, identified enhanced glycolysis metabolism pathway. Antagonizing ERβ in cell lines and in patient derived xenografts (PDX) demonstrated that ERβ is a therapeutical target in breast cancer and can be utilized to specifically target the CSC population. Tamoxifen is an important therapy for ERα positive breast cancers, however around 30-40% of patients relapse during endocrine therapy2. To investigate the endocrine resistance from a cancer stem cell perspective (Paper III), we treated adherent breast cancer cells (ER+) and CSCs with tamoxifen. Interestingly, CSCs where found to be resistant to tamoxifen treatment, while tamoxifen inhibited the adherent cancer cell population. To understand the mechanism behind the CSC induced endocrine resistance, we performed microarray analysis on patient derived CSCs treated with tamoxifen. Interestingly, mTOR signaling related pathways were found to be induced by tamoxifen in CSCs. This induction of mTOR effector downstream targets were observed only in CSCs but not in adherent cancer cells. Further, mTOR signaling was also found to be elevated in CSCs compared to the adherent cancer cell population. mTOR inhibitors such as rapamycin and everolimus were found to be effective in reducing the mammosphere formation. Therefore, combined tamoxifen and mTOR inhibitors can effectively target both differentiated cancer cells and the CSC population. Next, we explored the genomic landscape of metastases, patterns of metastatic spread and the role of axillary lymph node metastasis in seeding distant metastasis (Paper IV). We performed whole exome sequencing on 99 tumor samples from 20 breast cancer patients with matched primary and metastatic lesions. We observed both linear progression (i.e. metastasis seeding successive distant metastasis) and parallel progression (i.e. different distant metastasis were seeded from primary tumor directly rather than seeded by other distant metastasis) model during breast cancer progression. Majority of the distant metastasis where polyclonally seeded. We observed lack of axillary lymph node involvement in seeding distant metastasis. This indicates that, the majority of cancer cells are seeded hematogenously rather than utilizing the lymphatic system for cancer spreading. On average, only half of primary mutations were retained in the distant metastatic lesions with considerable disparity between individual patients ranging from 9 to 88%. Several putative driver alterations occurred late, privately in distant metastasis, highlighting the need to characterize the genomic alterations of metastatic lesions for making better informed clinical decision at metastatic setting. Further, we also observed specific mutational signatures such as APOBEC-associated signature, were significantly higher in distant metastasis compared to their respective primary tumors. Finally, in paper V, we profiled (RNA sequencing) multiple regions of the same tumor from 12 breast cancers. Molecular subtypes and transcriptomic grades for each tumor piece was determined. Primary breast cancers exhibited substantial intra-tumor genomic heterogeneity, but limited transcriptomic heterogeneity at macroscopic level. Our data suggested that, intra-tumoural heterogeneity is unlikely to have an impact on transcription based molecular diagnostics for most patients. In conclusion, we have identified potential therapeutic targets such as ERβ and mTOR pathway for inhibiting CSCs. Drugs targeting both CSCs and differentiated cancer cells are promising strategies to eradicate cancer recurrences. More clinical trials involving cancer stem cell targeting agents along with traditional therapies are required to investigate their clinical efficacy. Further, genomic characterization of both primary tumors and metastatic lesions are crucial for improving the treatment outcome for advanced breast cancer patients.