Recent studies indicate a correlation between high expression of the POZ-ZF transcription factor Kaiso, and the aggressiveness of the triple negative breast cancer (TNBC) subtype. However, little is known about the biological roles of Kaiso in TNBC tumorigenesis and metastasis, which laid the foundation for this thesis. To elucidate Kaiso’s role in TNBC, we generated stable Kaiso depletion in two well-established TNBC cell lines – MDA-MB-231 and Hs578T – using RNA interference technology. Intriguingly, we observed that Kaiso depletion delayed the tumor onset of MDA-MB-231 but not Hs578T cells, and led to the reduced expression of the c-Myc oncoprotein in MDA-MB-231 but not Hs578T cells. We postulate that this reduction in c-Myc expression is partly responsible for the delayed tumor onset observed in MDA-MB-231 cells. Additionally, loss of Kaiso expression resulted in increased apoptosis of both MDA-MB-231 and Hs578T cells in vitro and in vivo, which was accompanied by reduced expression of the DNA repair protein BRCA1. Remarkably, bioinformatic analysis revealed that high Kaiso and BRCA1 mRNA expression correlates with the reduced survival rates of TNBC patients. Further characterization of the Kaiso-depleted cells revealed that loss of Kaiso expression strongly inhibited the metastatic abilities of MDA-MB-231 and Hs578T cells. Importantly, Kaiso depletion led to decreased TGFβ-receptor I and II (TGFβRI and II) expression that is essential for the activation of the TGFβ signaling cascade. Concomitantly, suppressing Kaiso led to reduced TGFβ signaling. As increased TGFβRI expression is independently associated with the poor prognostic outcome of breast tumors, and the TGFβ signaling pathway is highly involved in breast tumor metastasis, we hypothesize that Kaiso functions together with TGFβRI and the TGFβ signaling cascade to promote TNBC metastasis. An additional goal of this thesis was to investigate the role of Kaiso in the prevalence of TNBC in women of African ancestry (WAA) compared to Caucasian women – since increased Kaiso expression is implicated in the poor survival outcomes of breast cancer patients of African ancestry relative to their Caucasian counterparts. Using tissue microarray and immunohistochemical analyses, we revealed for the first time a high nuclear expression of Kaiso in TNBC tissues of WAA (Nigerian, Barbadian, African American) compared to TNBC tissues of Caucasian women. Collectively, these findings unveiled functional oncogenic roles for Kaiso in the tumorigenesis and metastasis of TNBC, and revealed a plausible link between high Kaiso expression, high African ancestry and the predisposition of young WAA to TNBC. / Thesis / Doctor of Philosophy (PhD)

Breast cancer is a complex heterogenous disease that consists of several different subtypes displaying distinct behaviors and responses to different treatments. It is the second leading cause of cancer death among women, and is the most commonly diagnosed cancer in women. Although recent developments have helped shed light into this disease, there is still much to investigate. One particular subtype of breast cancer, known as triple-negative breast cancer, remains the most aggressive, as this tumor type is of high histological grade and preferentially affects women with BRCA1 mutations and women who are younger than 40 years of age. Unlike other subtypes with better prognoses, triple-negative breast cancer still has no targeted therapy, and chemotherapy remains the primary systemic treatment. Recently, there has been an increase of interest in the SOXC family of high mobility group transcription factors and their roles in tumor development. Studies have revealed some of the effects that SOXC genes may have on various tumor types. However, further studies are still needed to elucidate the roles, functions, regulations, and mechanisms of these transcription factors. This study aims to focus on one particular gene in the SOXC family known as sex determining region Y-box 11. Recent studies have shown that sex determining region Y-box 11, also known as SOX11, is one of the factors required for maintaining the basal-like breast cancer phenotype and is also critical in regulating growth, migration, invasion, and expression of signature basal-like breast cancer genes. Emerging evidence also reveals that this transcription factor may have an impact on homologous recombination repair when DNA damage occurs, in triple-negative breast cancer. Using SOX11 overexpression and knockout cell models combined with basic science laboratory techniques and omics, the next generation of laboratory tools, this study seeks to explore the role and function of SOX11 in DNA damage in triple-negative breast cancer. The results of this study have confirmed the recent findings of the role of SOX11 in cell proliferation and growth in triple-negative breast cancer. It has also revealed that overexpression of SOX11 in triple-negative breast cancer cell lines leads to an increase in DNA damage, loss of BRCA1 function, and dysregulation in the cell cycle. High expression of SOX11 is also associated with worse prognostic outcomes in triple-negative breast cancer patients. Because overexpression of SOX11 resulted in a loss of BRCA1 function, there may be a potential role for SOX11 in inducing the BRCAness phenotype commonly seen in basal-like breast cancers. The results of this study strongly suggest that SOX11 is involved in defective DNA damage repair pathways. Further studies need to be conducted in order to evaluate SOX11 as an inducer of the BRCAness phenotype, which occurs when there is a homologous recombination repair defect and no germline BRCA1 mutation present. Because of this, SOX11 may also have the potential to act as a functional biomarker for therapies targeting DNA damage, as recent developments in identifying therapies that could potentially target homologous recombination repair defects have been promising.

The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases, and loss of RAB 25 expression has been reported in a number of breast cancer cases containing H-Ras point mutations, particularly triple negative breast cancers (TNBC), one of the most aggressive subtypes of breast cancer and associated with a poor prognosis. The mechanism involved in the progression of these tumors is poorly understood. In this study, we are trying to understand if loss of RAB25 expression in Human Mammary Epithelial Cell (HMEC) lines co-operates with H-Ras mutations and contributes to tumorigenesis. HMEC were immortalized by transduction with LXSN CDK4 R24C, a mutant form of cyclin-dependent kinase, followed by transduction with hTERT, catalytic subunit of the telomerase enzyme that permits the cells to exceed the Hayflick Limit and become immortal. We have found that with loss of RAB25 and over expression of mutant H-Ras61L, immortal HMEC undergo transformation. We have looked into the co-operativity between loss of Rab25 and H-Ras61L mutant by in-vitro studies to show their anchorage independent growth and increased ability to migrate. Furthermore, cells express low CD24, high CD44, and very low levels of Claudin indicating that cells acquire stem-like properties upon transformation. Loss of RAB25 and over-expression of H-ras61L resulted in increased expression of transcription markers Snail and Slug that drive these cells to lose E-cadherin and undergo Epithelial Mesenchymal Transition (EMT). This study shows that loss of RAB25 and over-expression of mutant H-Ras can transform HMEC and give rise to mesenchymal stem-like tumors. Our findings reveal that RAB25 functions as a tumor suppressor gene, and loss of RAB25 could serve as a novel biomarker in the prognosis of Claudin-low type of TNBC.

A significant racial disparity in breast cancer mortality exists among women in the United States. Triple-negative breast cancer (TNBC) is a breast cancer phenotype that may explain, in part, this disparity between white and African American women. The objective of this study was to determine the predictors of survival in TNBC and non-triple-negative breast cancer (NTNBC) patients. Data on 168,756 female patients with a diagnosis of invasive breast cancer in the Surveillance Epidemiology and End Results (SEER) program were stratified based on breast cancer receptor phenotypes in this retrospective longitudinal cohort study design. Multiple logistic regressions were used for exploring predictors of treatment which showed that not receiving surgery as standard treatment was associated (odds ratio: 95% CI) with TNBC (OR 1.151: 1.042, 1.177), uninsured (OR 3.552: 3.206, 3.937) and African American (OR 1.804: 1.702, 1.912) while not receiving radiation was associated with TNBC (OR 1.151: 1.113, 1.190), uninsured (OR 1.318; 1.217, 1.429). Cox’s hazard models were used, regressing age, race, ethnicity, marital status, health insurance status, histological tumor grade, and treatment status on survival time, the outcome measure. Analysis revealed that the mean survival time is lower for TNBC [15.60 (± 10.29)] months compared with NTNBC [16.01 (± 10.18)] (p < 0.0001), a difference though small is statistically significant. The independent determinants of survival in TNBC were: young age at diagnosis [(β = 0.033, HR 1.033 (1.026, 1.041)]; being African American [(β = 0.182, HR 1.200 (1.117, 1.289)], being married [(β = - 0.362, HR 0.697 (0.658, 0.737)]; higher tumor histological grades [β = 1.034, HR 2.812 (2.159,3.661)]; uninsured [(β = 0.541, HR 1.717 (1.481, 1.992)]; no surgery [(β = 2.156, HR 8.633 (8.152, 9.143)], or no radiation treatment [(β = 0.489, HR 1.630 (1.535,1.73)]. African American race, uninsured status, higher grade at diagnosis, inadequate treatment are independent predictors of poor survival among breast cancer patients; importantly, TNBC had a lower survival than that of NTNBC patients. A higher proportion of TNBC patients had a diagnosis at younger age, with higher tumor grade and was of the African American race. The survival disparity in African American patients may be partially explained by disproportionately higher TNBC cases among them, as well as, rates of not receiving standard treatments.

archives@tulane.edu / Triple negative breast cancers (TNBCs) are clinically and biologically aggressive, with higher recurrence and metastasis rates compared to other subtypes. Acquisition of a mesenchymal and migratory cell phenotype is consequential process that promotes metastasis. There are no clinically approved small molecule targeted therapies for TNBC; kinases are effective drug targets in cancer research. Although some kinases are known to regulate the mesenchymal phenotype, a large subset of the human kinome is understudied. There are many approaches to discovering novel kinase targets in cancer. Here, a phenotypic screen approach is described to identify understudied kinases using the Published Kinase Inhibitor Set (PKIS). Initial screens using TNBC cell lines (MDA-MB-231, BT549 and MDA-MB-157) identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology. Our hits were further prioritized based on gene expression changes of the epithelial marker E-cadherin and migratory behavior. Active compounds were confirmed to reverse EMT on transcript and protein levels with qRT-PCR and Western blot. When pharmacologically similar compounds were more closely examined, different effects on cancer biology were observed (‘active’ versus ‘inactive’ compounds). Based on these observations, a kinase array was employed to compare both the active and inactive compounds to demonstrate how to identify candidate kinases responsible for the EMT reversal. Using this screening approach, small molecule inhibitors from the PKIS library (GSK346294A, GSK448459A, GSK237700A) were identified that were pharmacologically similar that reversed the mesenchymal phenotype in TNBC. These compounds have different biological effects in TNBC, despite having similar pharmacophores. Differential effects of the PKIS compounds on transwell migration, gene (qRT-PCR) and protein (Western blot) expressions, and mammosphere formation in TNBC cells was observed. In follow-up in vivo studies, our most active compound (GSK346294A) suppressed tumorigenesis and metastasis. RNA-sequencing confirmed downregulated pathways induced by GSK346294A treatment in TNBC cells included EMT, cytoskeletal rearrangement and cell cycle regulation. Because these compounds have different off-target activities, this approach can be used to identify candidate unique kinases responsible for the observed effects. NEK5 was one of these kinases candidates. NEK5 function remains understudied in cancer, and even more understudied in breast cancer. This study is the first, to our knowledge, to describe the function of NEK5 in breast cancer, specifically its roles in acquisition of a mesenchymal and migratory cell phenotype. Overexpression of NEK5 promotes a migratory and mesenchymal phenotype, and knockdown with a shRNA construct suppresses this migratory behavior. Data obtained using both qRT-PCR of the knockdown and overexpression cell lines, and follow-up RNA sequencing, revealed NEK5 regulates the PLAU/PAI-1/SRC axis. Furthermore, a role for NEK5 in resistance to SRC-targeting anticancer agents is demonstrated. The work described here demonstrates the utility of a novel approach to identify understudied kinases in cancer, and characterization of these kinases has potential impact in other metastatic diseases not limited to breast cancer. / 1 / Margarite Matossian

Background and Objectives: Mesothelin, identified as a tumor-associated biomarker, is more often overexpressed in triple receptor-negative breast cancer (TNBC) than in common luminal breast tumor subtype or normal tissues. The objective of this systematic review is to determine the association between the expressions of mesothelin with survival outcomes in patients with TNBC. Methods We searched the following electronic databases: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials, PubMed, and Web of Science with no time or language restriction till May 19, 2016. Any prospective or retrospective longitudinal studies that investigate the prognosis of TNBC with mesothelin baseline measurement were selected. Two reviewers independently assessed every article for inclusion, extracted data, and assessed the methodological quality of every eligible trial. Pooled measures of associations were summarized with meta-analyses. Results and conclusions Among the 592 patients with TNBC included in the four eligible studies, 269 patients (45.4%) demonstrated mesothelin expression. For the primary outcome OS, we found the trend toward decreased survival for patients with mesothelin-positive TNBC than those without mesothelin expression. We also found that for long-term OS, the association was statistically significant (OR = 0.46; 95% CI= 0.30 to 0.73; P< 0.001). For the secondary outcomes, we found that mesothelin expression in patients with TNBC was associated with lower DFS and higher overall mortality than those without mesothelin expression. Despite the limitations of sample size, this present study shows a significant association between mesothelin expressions and survival outcomes in patients with TNBC. Patients with mesothelin-positive TNBC could benefit from mesothelin-targeted immunotherapies recently in the development. / Thesis / Master of Science (MSc) / It is unclear whether mesothelin expression in triple-negative breast cancer (TNBC) is an independent prognostic marker for survival. To the best of our knowledge, no systematic review or meta-analysis has ever been done on this topic. The present systematic review aims to evaluate the role of mesothelin as a prognostic marker for TNBC. The primary objective of this review is to synthesize available evidence on the association between the expression of mesothelin and overall survival (OS) of patients with TNBC. The secondary objectives include determining the relationship between the expression of mesothelin and disease-free survival (DFS), distant metastases, and mortality. Despite some limitations, this study shows a significant association between mesothelin expressions and long-term OS rate as well as DFS rate and mortality rate in patients with TNBC. Mesothelin has a prognostic significance for patients with mesothelin based on our findings. Patients with mesothelin-positive TNBC could benefit from mesothelin-targeted immunotherapies in development.

Triple negative breast cancer (TNBC) represents an aggressive subtype for which only chemotherapy is available. The RB1 tumour suppressor is frequently lost in human breast cancer, primarily in TNBC. Loss of RB1 deregulates the cell cycle and is thought to affect BC response to endocrine, radiation, and chemotherapy. However, the global chemosensitivity of Rb null BC is not known. Here I demonstrate that RB1-deficient TNBC cells are highly sensitive to radiation, and moderately sensitive to doxorubicin and methotrexate. However, loss of RB1 does not increase sensitivity to multiple other drugs. Moreover, a non-biased screen of 2 RB-deficient versus 2 RB-proficient lines with ~3500 drugs did not reveal any difference in sensitivity, but identified disulfiram as a potent drug, which compared favourably with current chemotherapeutics against TNBC. Disulfiram’s efficacy was validated against 13 human TNBC lines with an average IC50 of 300nM. IQGAP1 was identified as a potential target of disulfiram.

Triple negative breast cancer (TNBC) represents an aggressive subtype for which only chemotherapy is available. The RB1 tumour suppressor is frequently lost in human breast cancer, primarily in TNBC. Loss of RB1 deregulates the cell cycle and is thought to affect BC response to endocrine, radiation, and chemotherapy. However, the global chemosensitivity of Rb null BC is not known. Here I demonstrate that RB1-deficient TNBC cells are highly sensitive to radiation, and moderately sensitive to doxorubicin and methotrexate. However, loss of RB1 does not increase sensitivity to multiple other drugs. Moreover, a non-biased screen of 2 RB-deficient versus 2 RB-proficient lines with ~3500 drugs did not reveal any difference in sensitivity, but identified disulfiram as a potent drug, which compared favourably with current chemotherapeutics against TNBC. Disulfiram’s efficacy was validated against 13 human TNBC lines with an average IC50 of 300nM. IQGAP1 was identified as a potential target of disulfiram.

Breast cancer is one of the most common malignancy diagnosed in women and is the primary cause of cancer-related deaths in women worldwide. It is a heterogeneous group of diseases that have a different response, prognosis, and clinical outcomes. Estrogen, progesterone and HER2 negative breast cancer, known as triple negative breast cancer (TNBC), does not respond to hormonal therapy. Basal-like breast cancer (BLBC) has shorter overall survival rate among other subtypes. Tumors sharing both TNBC and BLBC are considered less responsive to currently available treatment. Chemoresistance to treatment has been a challenge in cancer biology and force investigation toward developing new targeted therapies, which selectively target specific subtypes. Sphingolipid metabolites have an important physiological role in determining cell fate. Sphingolipid metabolites, ceramide, sphingosine, and sphingosine-1-phosphate (S1P), are implicated in cancer. S1P exerts its functions via extracellular and intracellular targets. S1P synthesized inside the cell is exported outside and binds to G-protein coupled receptors, the sphingosine-1-phosphate receptors 1-5 (S1PR1-5). Although the intracellular function is not well defined, its suggested intracellular S1P promotes cell apoptosis. The S1P pathway has received great attention recently due its function in cell survival and death. This effect was reported to be concentration dependent. In this research, I focused on S1P effect on nine TNBC/BLBC cell lines. I examined the in-vitro effects of S1P on apoptosis, proliferation, and cytotoxicity in triple negative/ basal-like breast cancer cell lines. Moreover, I studied the co-administration of S1P with currently used chemotherapeutic agents in these cell lines. Data show that S1P can selectively induce cell death in TNBC/BLBC cell lines at a specific concentration. In this research, I found that the mechanism of cell death following treatment with different S1P concentrations was mainly due to apoptosis. Results show that S1P leads to cell shrinkage, rounding and detachment in the nine TNBC/BLBC cell lines. S1P combination with doxorubicin and docetaxel at different concentrations shows no beneficial effect of the combination compared to the chemotherapeuitc agent alone. In some cell lines, the combination showed a protective effect. Further studies are required to determine the mechanism by which S1P induces cell apoptosis, inhibits cell growth, and demonstrates lack of responsiveness in combination studies.

Colon and breast cancers are amongst the leading causes of cancer deaths in the United States, mostly attributed to metastasis and resistance to therapy. Hence, there is a critical need to identify novel biomarkers for effective prognosis and to design targeted therapies to combat the metastatic diseases. Loss of heterozygosity (LOH) at chromosome 18q and inactivation of the target gene, SMAD4, corresponds to resistance to the common chemotherapeutic agent, 5-fluorouracil (5-FU), in colon cancer. Our examination of the therapeutic resistance phenomenon in SMAD4-negative colon cancer cells with the three common agents revealed significant resistance to both 5-FU and irinotecan but not to oxaliplatin. We also followed up with the earlier findings from our group, which suggested that SMAD4 might interact with metastasis-promoting factors to suppress metastatic progression and render sensitivity to chemotherapy. Co-immunoprecipitation and mass spectrometry analysis revealed that SMAD4 interacts with and inhibits RICTOR, a component of mTORC2 that activates oncogenic AKT via phosphorylation at Serine 473. Overexpression of SMAD4, depletion of RICTOR, or inhibition of AKT signaling restores sensitivity to irinotecan in SMAD4-negative colon cancer cells in vitro. Furthermore, as expected pharmacological inhibition of AKT sensitizes these cells to irinotecan in vivo. Interestingly, high RICTOR/AKT expression correlates with worse survival in colon cancer patients, suggesting them as novel prognostic biomarkers and therapeutic targets. On the other hand, triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer due to lack of effective targeted therapies. Using miRNA expression profiling of a model for epithelial-mesenchymal transition in TNBC, we found suppression of miR-4417 during the progression from non-malignant to malignant stage. Furthermore, localization of miR-4417 to chromosome 1p36, a region corresponding to high frequency of LOH in multiple cancers and low-level expression in TNBC patients associated with poor overall survival is consistent with its likely role as a tumor suppressor. Interestingly, we found that overexpression of miR-4417 is sufficient to inhibit migration and tumorigenecity of TNBC cells in vitro. Overall, our findings suggest miR-4417 exerts a tumor-suppressive effect and could serve as a novel prognostic biomarker and therapeutic tool against TNBC. / 2021-02-20T00:00:00Z