Karuna Mittal

Multinucleated polyploidy drives resistance to Docetaxel chemotherapy in prostate cancer

Background:Docetaxel is the only FDA-approved first-line treatment for castration-resistant prostate cancer (CRPC) patients. Docetaxel treatment inevitably leads to tumour recurrence after an initial therapeutic response with generation of multinucleated polyploid (MP) cells. Here we investigated role of MP cells in clinical relapse of CRPC.Methods:Prostate cancer (PC-3) cells were treated with docetaxel (5 nM) for 3 days followed by a washout and samples were collected at close intervals over 35 days post drug washout. The tumorigenic potential of the giant MP cells was studied by implanting MP cells subcutaneously as tumour xenografts in nude mice.Results:Docetaxel-induced polyploid cells undergo mitotic slippage and eventually spawn mononucleated cells via asymmetric cell division or neosis. Both MP and cells derived from polyploid cells had increased survival signals, were positive for CD44 and were resistant to docetaxel chemotherapy. Although MP cells were tumorigenic in nude mice, these cells took a significantly longer time to form tumours compared with parent PC-3 cells.Conclusions:Generation of MP cells upon docetaxel therapy is an adaptive response of apoptosis-reluctant cells. These giant cells ultimately contribute to the generation of mononucleated aneuploid cells via neosis and may have a fundamental role precipitating clinical relapse and chemoresistance in CRPC.

Multi-institutional study of nuclear KIFC1 as a biomarker of poor prognosis in African American women with triple-negative breast cancer

Nuclear KIFC1 (nKIFC1) predicts worse outcomes in breast cancer, but its prognostic value within racially distinct triple-negative breast cancer (TNBC) patients is unknown. Thus, nKIFC1 expression was assessed by immunohistochemistry in 163 African American (AA) and 144 White TNBC tissue microarrays (TMAs) pooled from four hospitals. nKIFC1 correlated significantly with Ki67 in White TNBCs but not in AA TNBCs, suggesting that nKIFC1 is not merely a surrogate for proliferation in AA TNBCs. High nKIFC1 weighted index (WI) was associated with significantly worse overall survival (OS), progression-free survival (PFS), and distant metastasis-free survival (DMFS) (Hazard Ratios [HRs] = 3.5, 3.1, and 3.8, respectively; P = 0.01, 0.009, and 0.007, respectively) in multivariable Cox models in AA TNBCs but not White TNBCs. Furthermore, KIFC1 knockdown more severely impaired migration in AA TNBC cells than White TNBC cells. Collectively, these data suggest that nKIFC1 WI an independent biomarker of poor prognosis in AA TNBC patients, potentially due to the necessity of KIFC1 for migration in AA TNBC cells.

Amplified centrosomes may underlie aggressive disease course in pancreatic ductal adenocarcinoma

Centrosome amplification (CA), the presence of centrosomes that are abnormally numerous or enlarged, is a well-established driver of tumor initiation and progression associated with poor prognosis across a diversity of malignancies. Pancreatic ductal adenocarcinoma (PDAC) carries one of the most dismal prognoses of all cancer types. A majority of these tumors are characterized by numerical and structural centrosomal aberrations, but it is unknown how CA contributes to the disease and patient outcomes. In this study, we sought to determine whether CA was associated with worse clinical outcomes, poor prognostic indicators, markers of epithelial-mesenchymal transition (EMT), and ethnicity in PDAC. We also evaluated whether CA could precipitate more aggressive phenotypes in a panel of cultured PDAC cell lines. Using publicly available microarray data, we found that increased expression of genes whose dysregulation promotes CA was associated with worse overall survival and increased EMT marker expression in PDAC. Quantitative analysis of centrosomal profiles in PDAC cell lines and tissue sections uncovered varying levels of CA, and the expression of CA markers was associated with the expression of EMT markers. We induced CA in PDAC cells and found that CA empowered them with enhanced invasive and migratory capabilities. In addition, we discovered that PDACs from African American (AA) patients exhibited a greater extent of both numerical and structural CA than PDACs from European American (EA) patients. Taken together, these findings suggest that CA may fuel a more aggressive disease course in PDAC patients.

Abstract B05: Centrosomal profiles of pancreatic adenocarcinoma from African American and European American patients: A comparative analysis

Background: Mortality from pancreatic cancer is higher in African-Americans (AA) compared to European Americans (EA). Although recent studies have focused on identification of gene expression signatures in whole tumors with variable metastatic potential, a disparity at the organelle-level between tumors of differing aggressiveness and metastatic potential has remained unexplored. Given the long-standing association between centrosome amplification (CA) and aggressiveness, we hypothesized that AA have a higher incidence and severity of CA in pancreatic adenocarcinomas as compared to EA. To test this hypothesis, we have developed an innovative method to quantitate the degree of CA (both numeral and structural) within tumor samples and defined a measurable index called the Centrosome Amplification Score (CAS). Experimental Design: Tissue specimens from 39 EA and 29 AA pancreatic adenocarcinomas were immunostained for centrosomes (gamma-tubulin) and nuclei (Hoechst). Immunofluorescence confocal imaging was then used to stack/take optical sections of tumor tissue and capture all centrosomes and nuclei within 10 regions of interest (ROIs) per sample. Centrosomes were categorized as (i) individually-distinguishable centrosomes (iCTRs) or (ii) as mega centrosomes (mCTRs) comprised of several tightly clustered centrosomes whose precise number could not be determined. For each ROI, the number of nuclei as well as the numbers and volumes of iCTRs and mCTRs were determined. The cumulative CAS was obtained for each ROI as CAS total = CASi + CASm (CASi - CAS for iCTRs and CASm - for mCTRs). Results: AA tumors (n=22) exhibit higher mean CASi (16.75 vs 13.6) and CASTotal (26.09 vs 22.48) than grade-matched EA (n=26) suggesting racial disparity at the organellar level (p Conclusion: Our results suggest that an aggressive disease course in AA can be attributed to higher degree and severity of CA (in particular numerical amplification) as compared to EA patients and may render AA more sensitive to centrosome targeting/declustering drugs. Our data compellingly suggest the potential usefulness of CAS in enabling patient stratification to channel patients into optimal treatment regimens with an overall goal of eliminating ethnic disparities in pancreatic cancer outcomes. Citation Format: Karuna Mittal, Vaishali Pannu, Padmashree C.G. Rida, Sergy Klimov, Sanam Sahjram Dharma, Helina w. Ketema, Michelle Dian Reid, Ritu Aneja. Centrosomal profiles of pancreatic adenocarcinoma from African American and European American patients: A comparative analysis. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B05.

Abstract PR02: β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor

Background: Androgen receptor (AR) has emerged as a new target for treating TNBC. AR is expressed in 10-43% of TNBCs. Although there are conflicting reports in the literature about the effect of AR status on TNBC prognosis, agents targeting AR signaling (enzalutamide) are already being evaluated in AR-positive TNBCs in early-stage clinical trials. However, no study so far has evaluated the association/correlation of AR status with ethnicity in TNBCs and downstream effects of AR loss in TNBCs. Given the association of AR loss with poor prognosis in breast cancer and that the African American (AA) with TNBC suffers aggressive disease course when compared to European American (EA) TNBCs, we hypothesized that AR loss might be an underlying cause of aggressive disease course in AR-negative TNBCs. Thus, in this project we aimed to study if loss or gain of AR in AA and EA TNBCs regulates the expression of β-catenin and leads to more aggressive disease course by activating downstream canonical Wnt-beta catenin signaling. Methods: We evaluated AR expression immunohistochemically in 424 formalin-fixed, paraffin-embedded samples from TNBC patients for whom complete clinicopathologic and overall survival (OS) data were available. Samples with Results: IHC staining of AR indicated that 79.5% of AA TNBCs (n=214) and 70% of EA TNBCs (n=210) were AR negative. Loss of AR was associated with poor overall survival in adjuvant-treated high Ki67 (>14%) (HR=1.72; p=0.095) AA TNBC (n=98) when compared to EA TNBCs (n=80). These data were validated by our in silico findings, which suggested that EA TNBCs (n=81) exhibited higher levels of AR mRNA compared to AA TNBCs (n=41) (p Conclusion: This study suggests that increased expression of β-catenin coupled with AR loss in AAs may underlie the ethnic disparity in outcomes among TNBC patients and strongly supports the prognostic role of AR and β-catenin in this breast cancer subtype. Citation Format: Karuna Mittal, Shristi Bhattarai, Sergey Klimov, Uma Krishnamurthi, Xiaoxian Li, Ceyda Sonmez Wetherilt, Mohammad A. Aleskandaran, Andrew A. Green, Emad A. Rakha, Ian O. Ellis, Guilherme Cantuaria, Guanhao Wei, Remus Mihai Osan, Meenakshi V. Gupta, Upender Manne, Padmashree C.G Rida, Ritu Aneja. β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr PR02.

Amplified centrosomes and mitotic index display poor concordance between patient tumors and cultured cancer cells

Centrosome aberrations (CA) and abnormal mitoses are considered beacons of malignancy. Cancer cell doubling times in patient tumors are longer than in cultures, but differences in CA between tumors and cultured cells are uncharacterized. We compare mitoses and CA in patient tumors, xenografts, and tumor cell lines. We find that mitoses are rare in patient tumors compared with xenografts and cell lines. Contrastingly, CA is more extensive in patient tumors and xenografts (~35–50% cells) than cell lines (~5–15%), although CA declines in patient-derived tumor cells over time. Intratumoral hypoxia may explain elevated CA in vivo because exposure of cultured cells to hypoxia or mimicking hypoxia pharmacologically or genetically increases CA, and HIF-1α and hypoxic gene signature expression correlate with CA and centrosomal gene signature expression in breast tumors. These results highlight the importance of utilizing low-passage-number patient-derived cell lines in studying CA to more faithfully recapitulate in vivo cellular phenotypes.

Abstract PR13: Disparities in centrosomal profiles: Prediction of metastatic risk in African American and European American breast cancer patients

Centrosome amplification has long been established as a hallmark of cancer. More than 80% of invasive breast tumors display this cellular trait. Centrosomal aberrations underlie chromosome instability (CIN) thus corroborating the extensive clonal intratumoral heterogeneity existent within breast lesions that fuels tumor evolution. Sub-clonal heterogeneity drives emergence of aggressive clones with a propensity to migrate and invade, resulting in tumor dissemination and metastases. Thus, centrosome amplification is a critical driver of tumor progression and metastases. Although numerous studies have linked centrosomal overload to tumor aggressiveness, no studies have yet quantified this cell-biological feature to establish a well-defined relationship between the severity and extent of centrosome amplification and tumor aggressiveness. Utilizing an innovative and rationally-guided approach, we have derived an algorithm that allows the precise quantitation of the frequency and severity of both structural and numerical aberrations in supernumerary centrosomes present in clinical samples. Our novel method thus uncovers previously unrecognized differences in the centrosomal profiles of grade-matched breast tumors from African-American (AA) (n=71) and European American (EA) (n=104) women. Our data demonstrate that AA breast tumors exhibit higher numeral, structural and total centrosome amplification scores than grade-matched EA tumors. Interestingly, tumors displaying lymph node and distant metastasis exhibited higher structural amplification than grade-matched non-metastatic tumors. Hence, our novel quantification tool offers valuable information that can potentially predict the risk of AA breast tumors rapidly progressing to metastatic disease and uncovers a hitherto unappreciated organelle-specific disparity marker among racially distinct breast tumors. This abstract was also presented as Poster B6. Citation Format: Nikita Wright, Vaishali Pannu, Padmashree Rida, Karuna Mittal, Sergey Klimov, Farida N. Yada, Michelle D. Reid, Guilherme Cantuaria, Ritu Aneja. Disparities in centrosomal profiles: Prediction of metastatic risk in African American and European American breast cancer patients. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr PR13.