Breast cancer progression is modulated by inherited genetic variance in multiple independent cohorts

Abstract

Cancer metastasis remains a major health issue and it is the main cause of cancer related mortality, responsible for an estimated 90% of solid tumour related deaths. Further research into the elements that contribute to cancer metastasis is therefore important in identifying key molecular factors and pathways modulating this complex process. This would subsequently enhance the understanding of the metastatic process and facilitate development and deployment of new and improved clinical management for patients with metastatic diseases.

Genetic sequence variation is one of the fundamental elements influencing the development of cancer and cancer metastasis. Evidence suggests that inherited genetic variations are one of the essential factors regulating breast cancer metastatic susceptibility; different inbred mouse strains with inherited polymorphisms have wide ranges of transgene-induced mammary tumour metastatic potency, with inherited genetic polymorphisms believed to give rise to a diverse range of metastatic susceptibilities. Molecular analyses, bioinformatics research and literature review have identified a number of potential breast cancer metastasis susceptibility modulating genes.

This study extended the research of potential metastasis modulator genes and investigated these genes in human populations, aiming to elucidate the connection of the inherited genetic nature of these potential breast cancer metastasis modulators and breast cancer patients. The hypothesis of this research is that the inherited genetic variance of these putative breast cancer metastasis susceptibility regulating genes, which were identified in previous mouse model research, are predictive of breast cancer patient diagnosis and prognosis. The identification of correlations between inherited genetic variation within novel breast cancer susceptibility genes, and clinical outcome would provide evidence supporting these genes as novel metastasis modulating genes.

ABI genotyping assays kits were utilized to perform SNP analysis of potential metastasis modulating gene variants in eight independent population cohorts (mostly Caucasian). This research first screened 49 candidate SNPs from 16 potential metastasis susceptibility modulating genes across two pilot cohorts that were chosen based on mouse model study results. Seven candidate SNPs from four different genes (SIPA1, ARAP3, RRP1B, BRD4) showed association with cancer survival in two pilot cohorts. These SNPs were subsequently assayed in other larger independent cohorts.

The results showed that two ARAP3 gene SNPs, rs440279 and rs3763120, were associated with important clinical markers - response to first line chemotherapy and reduced patient survival (with and without stratification of lymph node metastasis status and oestrogen receptor status). Four SIPA1 gene SNPs (SNP rs931127, rs746429, rs2448490 and rs3741378) were also found to be associated with patient outcome; rs746429 (missense SNP) was associated with poor outcome in metastasis-free, disease-free and overall-survival in the largest (Rotterdam, the Netherlands) population. The SNP rs2448490 (intronic SNP) from the same gene was found to be associated with better survival rate in the lymph node metastasis negative/oestrogen receptor positive patients. Since the SIPA1 protein and the RRP1B protein were shown to physically interact with each other and the SIPA1 SNP rs2448490 and RRP1B SNP rs9306160 both showed association with survival in the lymph node metastasis negative/oestrogen receptor positive class of patients; a combined analysis of the SIPA1 SNP rs2448490 and RRP1B SNP rs9306160 was performed, giving the best prognosis in the lymph node metastasis negative/oestrogen receptor positive subgroup.

SNPs in the other SIPA1 protein binding partner, BRD4, also showed association with progression-free survival. SNPs from the three genes, SIPA1, RRP1B and BRD4 which are known to be the corner-stone of the Diasporin pathway (Crawford, et al., 2008), a novel pathway in the metastasis process, showed association with breast cancer metastasis and metastasis related survival in multiple independent populations, adding support for these genes as metastasis modulator genes. In addition, the SNP rs3741378 from the SIPA1 gene also showed association with breast cancer incidence, indicating that the SIPA1 gene may not only be a breast cancer metastasis modulating gene but also a breast cancer susceptibility gene. To further investigate the role of this gene in breast cancer, molecular characterisation of an identified Sipa1 protein binding partner, Calmodulin 2, was also undertaken as part of this research. Calm2 was first identified as a potential Sipa1 protein binding partner from yeast two hybrid analysis, (Myriad Genetics, Salt Lake City, UT) with confirmation of binding verified by Coimmunoprecipitation assay. Stable cell lines with up-regulation of Calm2 gene were subsequently created. The stable cell lines were used in in vivo analyses to investigate the possible role of the Calm2 in breast cancer metastasis. However, deriving any firm conclusions from the in vivo analysis of Calm2 can’t be drawn as the control samples of this study didn’t provide adequate data and further investigations of Calm2’s possible role in metastasis are required.

These novel metastasis susceptibility modulating genes have previously shown association with breast cancer at the transcriptional and translational level. The results of this study provide evidence that these genes are engaged in human breast cancer survival at the genetic level. The data provide evidence of association between subtle inherited genetic variation of these novel breast cancer metastasis modulators and breast cancer patient outcomes in various independent human populations. The results of this research also provide additional evidence that genetic variation is involved in human breast cancer metastasis and survival. The observation that genetic variation may be an independent indicator for lymph node metastasis, negative/oestrogen receptor positive status, suggests a novel hypothesis in relation to breast cancer biology, ie breast tumours that do and do not colonize to the lymph node system may represent two distinct subtypes of breast cancer, with distinct tumour cell biology and mechanisms of progression.