Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer

Increased mitotic activity is a hallmark of aggressive cancer and is associated with genome instability, increased proliferative activity, and reduced patient survival in many types of cancer. In pursuit of mechanisms that increase mitotic activity in breast cancer, we identified a 54-gene mitotic apparatus network that is transcriptionally upregulated in primary tumors and breast cancer cell lines with high mitotic activity and/or high proliferative capacity. In parallel, we defined a mitotic network activity index (MNAI) as a quantitative measure of the transcriptional activity of the entire 54-gene network and showed that high MNAI is enriched amongst basal-like or IC10 tumors. We further showed that elevated MNAI is significantly associated with poor prognosis independent of standard clinical covariates.

Our data suggest that high MNAI and the elevated expression of the mitotic network genes can be explained, in part, by co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25, which encode the transcription factors MYC, ZEB1, FOXM1, and SOX9, respectively. Indeed, each of the 54 genes in the mitotic apparatus network have predicted binding sites for one or more of these transcription factors and we verified the majority of binding sites for MYC, FOXM1, ZEB1, and SOX9 using publicly available ChIP-seq datasets. A genomic mechanism of mitotic apparatus network activation in cancer is consistent with the observation that the transcriptional activity of the mitotic network appears to be under genetic control in normal tissues both in the mouse [15] and human lymphocytes. These amplified transcription factors are known to play important roles in normal tissue development and/or stem cell biology. In particular, MYC has been implicated in reprogramming somatic cells to become pluripotent stem cells [37]. ZEB1 has been associated with the epithelial-to-mesenchymal transition and cell migration [38]. SOX9 has been implicated in neural crest tissue development, the maintenance of multipotency, and Notch-mediated cell fate determination [39, 40]. FOXM1 is a transcription factor implicated in mitosis, a component of the 54-gene mitotic network, and a known transcriptional target of estrogen receptor alpha, with an important role in breast cancer endocrine biology [41]. These diverse functions may explain why tumors with high genome amplification-associated MNAI also have increased invasive potential and take on features associated with stem cells. Several cell proliferation signatures have been previously reported and shown to be associated with poor prognosis in subsets of breast cancer patients [42, 43], and not surprisingly these gene sets overlap with the MNAI. For example, many of the genes in the 54-gene mitotic apparatus network are included in the CIN25 gene signature reported to be associated with genome instability and reduced survival in multiple tumor types [4]. The association with genomic instability may stem from the deregulation of multiple mitotic apparatus genes via co-amplification of key transcription factors that influence genome instability both directly by interfering with DNA repair and the mechanical aspects of chromosome segregation and indirectly by deregulating checkpoint genes that normally function to inhibit cell cycle progression in cells with mechanical or genomic aberrations. Gene ontology analysis of the 54-gene mitotic apparatus signature indicates that CCNB1, CENPE, DLGAP5, HJURP, KIF2C, NCAPD2, NCAPG, NCAPH, NDC80, PTTG1, and SMC4 are involved in mechanical aspects of chromosome segregation, whereas CHEK1, EXO1, PTTG1, RFC3, and TYMS are involved in DNA repair, and finally, BUB1, BUB1B, CCNA2, CCNB1, CENPE, CHEK1, GSTE1, PLK1, and TTK are cell cycle checkpoint genes.

Genome amplification-driven activation of the mitotic apparatus network raises the possibility that cancers with this mechanism of activation have become “addicted” to the activation and thus, will be more sensitive to agents that target the activated network proteins than tumors with lower activity. Consistent with this, we have shown that the small molecule inhibitors GSK462364, GSK923295, and GSK1070916 that target the network proteins PLK1, CENPE, and AURKB/C, respectively, inhibit the growth of breast cancer cell lines with high MNAI at lower concentrations than cell lines with low MNAI. These results also are supported by the report that treatment with the aurora kinase inhibitor, VX-680, selectively kills cells that over express MYC [44]. siRNA knockdown experiments show that inhibition of most genes in the mitotic apparatus network significantly represses growth, and implicate AURKA, TTK, MELK, and PBK as additional druggable proteins in the network. Based on our previous data (AACR 2008, Abstract# 2397) and others reported, these inhibitors not only induced the accumulation of cells with 4?N and ?4?N DNA content, suggesting that DNA replication could occur in the absence of cytokinesis, indicative of a cell-cycle block in either G2 phase or mitosis, but also induced apoptosis in human cancer cell lines. Although GSK1070916 has potent activity against proliferating cells, a dramatic shift in potency is observed in primary, non-dividing tumors [45]. These observations indicate that mitotic apparatus inhibitors might be best targeted to aggressive cancers with high MNAI and/or co-amplification of MYC, ZEB1, FOXM1, and SOX9, thereby lowering the dose required for effective treatment and correspondingly lowering overall toxicity. Our studies of pairwise combinations of GSK462364, GSK923295, and GSK1070916 show that toxicity does not appear to be additive. Thus, combinations of compounds targeting multiple mitotic apparatus proteins might be deployed either together or sequentially to counter therapeutic resistance. This approach might lead to more durable treatment of the most aggressive forms of breast cancer.