Abstract P1-01-23: Hypoxia induced centrosome amplification via HIF-1α/Plk4 signaling axis associates with poorer overall survival in TNBC
Authors: Mittal K , Choi DH , Maganti N , Ogden A , Melton BD , Kaur J , Gupta MV , Jonsdottir K , Janseen EAM , Aleskandarany MA , Rakha EA , Rida PCG , Aneja R 
Published: February 2018

Background: Centrosome amplification (CA) which refers to presence of supernumerary or abnormally large centrosomes drives tumor progression by promoting chromosomal instability and the generation of aggressive tumor clones. Although the role of CA in cancer progression is well-defined, no studies have yet discussed how CA is induced in tumor cells. We report here that intra-tumoral hypoxia, which is considered one of the major contributors to intratumor heterogeneity, induces CA via HIF-1α.

Methods: We first immunohistochemically labeled 24 breast carcinoma and uninvolved adjacent normal tissue samples for HIF-1α and calculated weighted indices (WIs) for nuclear HIF-1α. Adjacent serial sections from the same tumors were also immunofluorescently labeled for γ-tubulin and CA was calculated. Using public microarray datasets (Kao dataset, n=327), we investigated whether centrosomal gene expression is enriched in breast tumors characterized by a hypoxia gene expression signature. Finally, to determine the role of hypoxia in CA induction we exposed cultured TNBC cells (MDA-MB-231 and MDA-MB-468) to hypoxia and overexpressed (OE) and knocked out (KO) HIF-1α in TNBC cells and quantitated CA. Additionally, to discern the biological pathway through which HIF-1α induces CA we performed ChIP assay and in silico analyses to identify the possible targets of HIF-1α.

Results: A strong positive correlation between nuclear HIF-1α WI and CA was found in breast tumor samples (Spearman's rho p=0.722, p&lt;0.001). In addition, we found that higher nuclear HIF-1α was associated with worse overall survival (p=0.041; HR=1.03). Our in silico findings suggest that breast tumors with high expression of hypoxia-associated genes exhibited higher expression of centrosomal genes than breast tumors with low expression of hypoxia-associated genes. In addition, cells cultured in hypoxic conditions exhibited ˜1.5 fold higher (p&lt;0.05) CA when compared to the cells cultured in normoxic conditions. Interestingly level of CA decreased when HIF-1α KO TNBC cells were exposed to hypoxia and it increased when HIF-1α OE TNBC cells were culture in normoxic conditions. Furthermore, we discovered that HIF-1α induced CA by directly regulating the expression of Plk4 which was confirmed by performing ChIP assay. Our results indicated HIF-1α interaction with the motif in the PLK4 promoter from genomic DNA of MDA-MB 231 cells under hypoxic conditions, was significantly (p=0.04) higher when compared with the cells cultured under normoxic conditions. Plk4 mRNA expression was assessed using the online BC gene expression data sets (n=25). We found significantly higher expression of Plk4 in TNBC (n=374) when compared with non-TNBC (n=4098) and it was associated with poor overall survival (HR=1.76; p=0.054) in TNBC.

Conclusion: Collectively our findings suggest that hypoxia drives CA in TNBC via HIF-1α and contribute to poor outcomes. Thus, determination of CA and HIF-1α can help risk stratification in TNBC patients for more personalized treatments.

Citation Format: Mittal K, Choi DH, Maganti N, Ogden A, Melton BD, Kaur J, Gupta MV, Jonsdottir K, Janseen EAM, Aleskandarany MA, Rakha EA, Rida PCG, Aneja R. Hypoxia induced centrosome amplification via HIF-1α/Plk4 signaling axis associates with poorer overall survival in TNBC [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-23.