To Örebro University

Background: Overexpression of the human epidermal growth factor receptor (HER) 2 is associated with poor prognosis and shortened survival in breast cancer patients. HER2 is a potent activator of several signaling pathways that support cell survival, proliferation and metabolism. In HER2-positive breast cancer there are most likely unexplored proteins that act directly or indirectly downstream of well established pathways and take part in tumor development and treatment response.

Methods: In order to identify novel copy number variations (CNVs) in HER2-positive breast cancer whole-genome single nucleotide polymorphism (SNP) arrays were used. A PCR-based loss of heterozygosis (LOH) assay was conducted to verify presence of deletion in HER2-positive breast cancer cases but also in HER2 negative breast cancers, cervical cancers and lung cancers. Screening for mutations was performed using single-strand conformation polymorphism (SSCP) followed by PCR sequencing. Protein expression was evaluated with immunohistochemistry (IHC).

Results: A common deletion at chromosome Xq24 was found in 80% of the cases. This locus harbors the gene solute carrier (SLC) family 25A member 43 (SLC25A43) encoding for a mitochondrial transport protein. The LOH assay revealed presence of SLC25A43 deletion in HER2-positive (48%), HER2-negative (9%), cervical (42%) and lung (67%) cancers. HER2-positive tumors with negative or low SLC25A43 protein expression had significantly lower S-phase fraction compared to tumors with medium or high expression (P = 0.024).

Conclusions: We have found deletion in the SLC25A43 gene to be a common event in HER2-positive breast cancer as well as in other cancers. In addition, the SLC25A43 protein expression was shown to be related to S-phase fraction in HER2-positive breast cancer. Our results indicate a possible role of SLC25A43 in HER2-positive breast cancer and support the hypothesis of altered mitochondrial function in cancer.

Solute carrier family 25A member 43 (SLC25A43) gene is a putative tumor suppressor gene that undergoes loss of heterozygosity (LOH) in human epidermal growth factor receptor 2 (HER2) positive breast cancer. Also, knockdown of SLC25A43 in cell lines influences cell turnover and metabolism. Absence of mutations in this gene in breast cancers prompted us to study methylation as an alternate mechanism for gene inactivation of this X encoded gene. Quantification of CpG site methylation using pyrosequencing was performed upstream of the SLC25A43 gene and at its 5' end in a cohort of breast tumor tissues (n = 80, HER2 positive or negative) with different SLC25A43 gene deletion status. Compared with control tissue, cancer tissues had lower levels of methylation at the 5' and 3' shores of the gene. Cancer tissues with no deletion in the SLC25A43 gene (Del(-)) had higher methylation in the CpG island (CGI) of the gene than cancers carrying the deletion (Del(+)). Methylation in the CGI of the SLC25A43 gene was negatively correlated with age at diagnosis. In HER2 positive breast cancer, ER negativity and lymph node positivity was associated with higher methylation in the CGI and in the adjacent shores of this gene. Our results suggest that methylation in the CGI of the SLC25A43 gene could be an alternate mechanism of gene silencing in the absence of LOH. Also, associations between site-specific methylation and clinicopathological parameters suggest that epigenetic changes in SLC25A43 gene could be of importance in breast carcinogenesis.

With the aim of obtaining a more comprehensive epigenetic signature in HER2-positive breast cancer (HER2+ breast cancer), we performed a genome-wide methylation analysis on 17 HER2+ breast cancer and compared to 10 normal breast tissue samples using the Illumina Infinium HumanMethylation450 BeadChip that interrogates >485,000 CpG loci per sample. Our findings show that altered DNA methylation, more specifically hypermethylation occur at CpG islands in HER2+ breast cancer affecting genes involved in multicellular development, differentiation and transcription, and was overrepresented by the homeobox family of genes, including those which have not been previously reported in relation to cancer (DBX1, NKX2- 6, SIX6). Alteration in methylation was also found to affect genes of the PI3K and the Wnt signalling pathways in HER2+ breast cancer. Notably among them are HER2, AKT3, HK1 and PFKP, in which altered methylation has not been previously reported. We have identified 73 candidate biomarker genes in HER2+ breast cancer and external validation of gene expression in a selected group of these genes (n=13) revealed lowered mean gene expression in HER2+ breast cancer. Future clinical studies focusing on these candidate biomarker genes as well as homeobox-containing genes and HER2, AKT3, HK1 and PFKP are warranted which could provide further insights to the biology of HER2+ breast cancer.

Introduction: Breast cancers with human epidermal growth factor receptor (HER) 2 gene amplification or protein overexpression (HER2+ breast cancer) is associated with poor prognosis. Activated HER2 receptors dimerise and activate the oncokinase Akt; which phosphorylate the tumour suppressor protein Forkhead box O3a (FOXO3a), repressing its transcriptional activity. Oncogenic FOXG1 can act as a transcriptional repressor for genes which are transcriptionally activated by FOXOs and phosphorylation of FOXG1 via Akt causes its nuclear export in differentiated cells. To better understand the AKT/FOXO3a/FOXG1 connection and their clinical relevance, we investigated the expression and localisation of pAkt, pFOXO3a and FOXG1 in HER2+ breast cancer.

Methods: Immunohistochemical analysis was performed to determine the expression and localisation of pAkt, pFOXO3a and FOXG1 on tissue microarray constructs from HER2+ primary breast tumours (n=91) and their relation to clinic pathological parameters were analysed.

Results: Nuclear expression of pAkt was found to be correlated to nuclear (p<0.001) as well as cytoplasmic expression of pFOXO3a (p = 0.006), while cytoplasmic expression of pAkt was found to be correlated to cytoplasmic expression of pFOXO3a (p<0.001). Nuclear expression of pFOXO3a was inversely correlated to cytoplasmic expression of FOXG1 (p=0.003). Cytoplasmic expression of pFOXO3a was found to be associated with sentinel node metastasis (p=0.011), while cytoplasmic FOXG1 expression was correlated to negative progesterone receptor status (p=0.008).

Conclusion: Our results suggest that the expression and localisation of pAkt and pFOXO3a is interconnected, while the expression of FOXG1 is connected to pFOXO3a. Our findings indicate the biological value of expression as well as localisation of these proteins in HER2+ breast cancer.