Abstract 3560: PLK4 is a HIF-1α target gene: A novel mechanism inducing centrosome amplification in tumor cells

Abstract

Abstract Background: The presence of supernumerary or abnormally large centrosomes, also known as centrosome amplification (CA), drives tumor progression by enhancing chromosomal instability, and altering cellular microtubule cytoskeletons and tissue cytoarchitectures to promote the migration and invasion capabilities of cancer cells. Although the prevalence of CA is documented in a variety of cancer types, it remains unclear how CA is induced in tumor cells.This study aims to examine the role of intratumoral hypoxia in inducing CA via HIF-1α. Methods: We (a) exposed cultured breast, pancreatic, colorectal and prostate cancer cells to hypoxia, and (b) genetically manipulated hypoxia [via overexpression (OE) or knock-out (KO) of HIF-1α], and quantitated CA employing immunocytofluorescence methods. We also quantitated the levels of CA-associated genes and proteins by qRT-PCR and western blot, respectively. Next, to discern the biological pathway through which HIF-1α induces CA, we performed ChIP and Luciferase reporter assays. We immunohistochemically labeled breast (n=24), colon (38), prostate (n=52), and pancreatic (n=34) cancer tissue samples for HIF-1α and PLK4. Serial sections from the same tumors were also immunofluorescently labeled for γ-tubulin and CA was quantitated. Using public microarray datasets (TCGA), we investigated whether centrosomal gene expression is enriched in tumors characterized by a high expression of a hypoxia gene expression signature. Results: Cells cultured in hypoxic conditions exhibited higher CA (p<0.05) than the cells cultured in normoxic conditions. CA levels increased when cells with HIF-1α OE were cultured in normoxic conditions and no significant induction of CA was observed when the HIF-1α KO cells were cultured under hypoxic conditions. ChIP and Luciferase assay confirmed that HIF-1α induced CA by directly upregulating the expression of PLK4. Moreover, when tumor cells were exposed to hypoxia after knock-down of PLK4, no significant changes in CA and CA-associated proteins were observed. In clinical patient samples (breast, colon, pancreatic and prostate cancer), we observed that HIF-1α expression was significantly (p<0.05) correlated with PLK4 expression at the protein level (breast ρ=0.81, colon ρ=0.76, prostate ρ=0.68, and pancreatic ρ=0.67). Findings from our in silico analyses further validated these observations by showing a strong correlation between the expressions of HIF-1α and PLK4 at the transcriptional level. We also found that centrosomal gene expression is enriched in tumors with high hypoxia score (TCGA dataset of breast, colon, pancreatic and prostate cancer). Conclusion: Collectively, our findings suggest that hypoxia drives CA in tumor cells through HIF-1α-mediated transcriptional upregulation of PLK4. Given the development of several promising HIF-1α inhibitors, this research could potentially aid in patient risk stratification for clinical decision-making, and enable design of new therapies. Citation Format: Karuna Mittal, Jaspreet Kaur, Shaligram Sharma, Ishita Choudhary, Guanhao Wei, Precious Imhansi Jacob, Da Hoon Choi, Nagini Maganti, Shrikant Pawar, Michael S. Toss, Emiel A. Janssen, Meenakshi V. Gupta, Michelle D. Reid, Emad A. Rakha, Padmashree Rida, Ritu Aneja. PLK4 is a HIF-1α target gene: A novel mechanism inducing centrosome amplification in tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3560.