Biological and therapeutic aspects of breast cancer progression

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

Intra-tumor heterogeneity in breast cancer has limited impact on transcriptomic-based molecular profiling

Background: Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. Methods: In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. Results: Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. Conclusions: Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.Peer reviewe

VKORC1 Pharmacogenetics and Pharmacoproteomics in Patients on Warfarin Anticoagulant Therapy: Transthyretin Precursor as a Potential Biomarker

Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke.We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation.This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism

Estrogen Receptor beta as a Therapeutic Target in Breast Cancer Stem Cells

Background: Breast cancer cells with tumor-initiating capabilities (BSCs) are considered to maintain tumor growth and govern metastasis. Hence, targeting BSCs will be crucial to achieve successful treatment of breast cancer. Methods: We characterized mammospheres derived from more than 40 cancer patients and two breast cancer cell lines for the expression of estrogen receptors (ERs) and stem cell markers. Mammosphere formation and proliferation assays were performed on cells from 19 cancer patients and five healthy individuals after incubation with ER-subtype selective ligands. Transcriptional analysis was performed to identify pathways activated in ER beta-stimulated mammospheres and verified using in vitro experiments. Xenograft models (n = 4 or 5 per group) were used to study the role of ERs during tumorigenesis. Results: We identified an absence of ERa but upregulation of ER beta in BSCs associated with phenotypic stem cell markers and responsible for the proliferative role of estrogens. Knockdown of ER beta caused a reduction of mammosphere formation in cell lines and in patient-derived cancer cells (40.7%, 26.8%, and 39.1%, respectively). Gene set enrichment analysis identified glycolysis-related pathways (false discovery rate amp;lt; 0.001) upregulated in ER beta-activated mammospheres. We observed that tamoxifen or fulvestrant alone was insufficient to block proliferation of patient-derived BSCs while this could be accomplished by a selective inhibitor of ER beta (PHTPP; 53.7% in luminal and 45.5% in triple-negative breast cancers). Furthermore, PHTPP reduced tumor initiation in two patient-derived xenografts (75.9% and 59.1% reduction in tumor volume, respectively) and potentiated tamoxifen-mediated inhibition of tumor growth in MCF7 xenografts. Conclusion: We identify ER beta as a mediator of estrogen action in BSCs and a novel target for endocrine therapy.Funding Agencies|Swedish Society of Medicine; Swedish Society for Medical Research (SSMF); Magnus Bergvalls Stiftelse; Karolinska Institutes Theme Center in Breast Cancer (BRECT); Linnecenter for Prevention of Breast and Prostate cancer (CRisP); Swedish Cancer Society; Stockholm Cancer Society; King Gustav V Jubilee Fund; Karolinska Institutet; Stockholm County Council Research Strategy Committee; Swedish Breast Cancer Association; Science for Life-Astra Zeneca; Marie Curie Actions via the VINNOVA program Mobility for Growth [291795]</p

The distribution of <i>VKORC1</i> diplotypes in patients receiving low-dose (N = 25) and high-dose (N = 28) warfarin.

<p>The distribution of <i>VKORC1</i> diplotypes in patients receiving low-dose (N = 25) and high-dose (N = 28) warfarin.</p

Effect of <i>VKORC1</i> diplotypes [H1H1 (n = 24), H1H7/H1H9 (n = 18) and H7H7/H7H8H9 (n = 9)] on (A) free T₃ (FT₃), (B) total T₃ (TT₃), (C) free T₄ (FT₄), and (D) total T₄ (TT₄) levels in patients receiving warfarin.

<p>Patients harboring the H1H1 diplotype group had significantly lower FT₃ and TT₃ levels compared with patients carrying the high dose (H1H7/H1H9) associated diplotype groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015064#pone-0015064-g003" target="_blank">Figures 3A and B</a>; P<0.05 in each case). These findings probably reflect the higher levels of TTR expressed in patients carrying the low dose H1H1 diplotype, which probably leads to lower levels of FT₃ as well as TT₃.</p

Immunoblot expressions of TTR levels in HepG2 cells exposed to low (1.0 µg/mL) and high (10 ug/mL) concentrations of warfarin for 24 hrs.

<p>TTR expression levels were determined in (A) serum-rich, and (B) serum-free conditions to exclude the possibility of false positive results. The data (mean ± S.E.) represents expression ratios of biological replicates normalized to GAPDH. The decrease in expression of TTR was significantly greater in HepG2 cells exposed to higher concentration (10 ug/mL) of warfarin under serum-rich conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015064#pone-0015064-g004" target="_blank">Fig. 4A</a>; P = 0.01).</p

Influence of <i>VKORC1</i> diplotypes on TTR precursor level in warfarin treated Asian patients (N = 51).

<p>Patients carrying the H1H1 diplotype showed significantly higher levels of TTR precursor compared to patients harboring the H1H7/H1H9 (P = 0.001) and H7H7/H7H8H9 diplotypes (P<0.0001).</p

Differential expression of plasma protein levels in patients treated at low- and high-dose warfarin identified by iTRAQ-coupled LC-MS/MS analysis.

<p>A total of 163 proteins were identified and 11 proteins with significant expression levels were identified based on ProtScore with a cut-off value of 2.0 at 99% confidence value. Of the eleven proteins, the median expression level of transthyretin precursor was highly significant between patients receiving low- and high-dose warfarin group (low-dose: 1.53, range: 0.828 to 3.83; and high-dose: 0.818, range: 0.534 to 1.483; P<0.0001).</p