In this manuscript, we identified novel genes and gene networks correlated with variable response to platinum-based chemotherapy in HGSOC patients. Using a univariate analysis approach, we identified a differentially expressed locus encoding the valosin-containing protein (VCP) associated with sensitivity to platinum-based chemotherapy. In addition, we applied a multivariate, co-expression analysis method for identifying groups of interconnected genes that could contribute to common biological pathways. This method identified 5 clusters of co-expressed genes nominally correlated with chemo-response, one of which remained significant after correction of multiple testing. Genes in these modules have been annotated to be associated with biological pathways such as apoptosis, transcription, immune response, negative regulation of the Wnt signaling pathway and DNA double-strand break processing involved in repair via single-strand annealing.
The most significantly associated probe identified in the DGE analysis was for a gene encoding Valosin-containing protein (VCP, p = 3.91E-06). We have confirmed that this signal is replicated in an independent ovarian cancer cohort with statistical significance (p = 0.015). VCP plays a critical role in disintegrating large polypeptide cellular structures for further degradation by proteolytic enzymes. It functions to regulate important pathways of DNA repair, replication and cell cycle progression by removing faulty polypeptide structures from chromatin material, ribosomes, endoplasmic reticulum and mitochondria. This gene has been previously identified as a potential biomarker for predicting the success of platinum-based chemotherapy in lung cancer patients.40
Many of the nominally correlated genes from our DGE analysis are enriched for pathways known to be critical for oncogenesis and chemotherapeutic drug resistance. For example, probe 213532_at mapping to the A Disintegrin and Metalloproteinase-17 (ADAM17) gene was upregulated in the chemo-resistant group (p = 0.017). This is consistent with prior studies, which reported that overexpression of ADAM17 results in reduced cisplatin-induced apoptosis in hepatocellular carcinoma cells and may contribute to cisplatin resistance via the EGFR pathway41. Moreover, we identified another up-regulated probe 205239_at (p = 0.003) for the gene encoding Amphiregulin (AREG), a protein found in the EGFR signaling pathway, which has been reported to promote ovarian cancer stemness and drug resistance to anti-cancer therapy.42 Taken together, our findings support a potential role of the AREG-EGFR signaling pathway in the development of platinum-resistance in ovarian carcinoma.
It is also important to note that one of the most enriched pathways from our DGE analysis was the protein processing in endoplasmic reticulum (hsa04141) pathway. Using DAVID, we have identified 18 differentially expressed probes mapping to 15 unique genes within this pathway. Prior studies have highlighted that endoplasmic reticulum (ER) stress may cause cisplatin resistance in ovarian carcinoma by inducing autophagy in cancer cells, allowing them to escape apoptosis.43,44 Findings from this pathway analysis suggest that future studies of the contribution of ER stress in cisplatin sensitivity is needed to improve our understanding of platinum-resistance in ovarian cancer.
In our co-expression network analysis, the gene module “honeydew1” showed the most significant correlation with chemotherapy response (p = 6.53e-05). This association signal was validated with statistical significance (p = 5.88e-07) in an independent replication cohort. This module includes two probes that map to VCP, a gene that was associated with platinum-based chemotherapy response in our DGE analysis. Additional genes in this module were associated with positive regulation of mitochondrial membrane potential, protein ubiquitination, mitosis, alternative splicing, and apoptotic processes. Pathway analysis showed that this module is involved in protein processing in the endoplasmic reticulum. A prior study has found that VCP plays a crucial role in cancer cell survival through extraction and degradation of unfolded proteins in endoplasmic reticulum, and noted that lower expression of VCP was associated with poor response to platinum-based chemotherapy.45 In alignment with this finding, genes co-expressed in the honeydew1 module were co-downregulated in chemo-resistant patients.
The honeydew1 module is composed of 76 probes mapping to 54 unique genes, and of these, 44 genes are located in chromosome 9, demonstrating the importance of chromosome 9 in the regulation of chemo-resistance in ovarian cancer. These findings support previous studies, where genetic imbalance and alterations in chromosome 9 have been associated with progression of ovarian cancer and increased cisplatin resistance.46 Analysis of overrepresented transcription factor binding sites demonstrated that genes in this module may be co-regulated by a common transcription factor known as organic cation transporter 1 (OCT1). We found that over 96% of genes in this module (49/54 genes) contain a nucleotide motif bound by OCT1. Prior studies have reported that silencing OCT1 impaired cisplatin-induced apoptosis in esophageal cancer cells, and that cisplatin-resistant cells were already expressing significantly reduced levels of OCT1.47 Taken together, these findings characterize a network of co-expressed genes that is associated with platinum-resistance in ovarian cancer. Genes within this module may be co-regulated by the OCT1 transcription factor, which may be used as a novel potential target for ovarian cancer therapies
The other four co-expression modules, which we were also able to replicate in an independent cohort, include genes known to be involved in oncogenic process and drug response outcomes. For example, the orangered4 module is composed of genes that are critical for regulation of immune response, which have been shown to play a role in chemotherapy response in HGSOC.14,15 Genes in this module are associated with functional annotation terms including immunoglobulin receptor binding, antigen binding, B cell receptor signaling pathway, and phagocytosis. In addition, 10 of the 58 genes in this module are enriched for a common transcription factor binding site: acute myeloid leukemia 1 protein (AML1). This transcription factor is involved in haematopoiesis process and immune functions such as thymic T-cell development. Studies have reported that the AML1 transcription factor is overexpressed in ovarian cancer patients, and may contribute to cancer cell proliferation, migration and invasion.48 In addition, we found that the lightpink3 module is strongly associated with transcription regulation process, which plays a pivotal role in cancer progression. Finally, genes in modules lightcyan1 and skyblue3 are target regions of chemotherapeutic agents such as tyrosine kinase inhibitors (Supplemental data 6).
Targeted analysis of BRCA1 and BRCA2 SNPs demonstrated that 28 out of 35 variants associated with chemotherapy response were also cis-acting eQTLs, correlated with the expression of BRCA2 as well as neighboring genes N4BP2L1, N4BP2L2, FRY, and STARD13 (nominal p-value < 0.05). Both BRCA2 and STARD13 are well known tumor-suppressors, and upregulation of N4BP2L1 and N4BP2L2 have been associated with positive prognosis in ovarian cancer cases.49 This finding shows the potential regulatory effect of the variants in BRCA2.
In addition, our annotation results show that 47% of platin-response associated variants in BRCA2 are linked with LDL/HDL cholesterol levels (Supplemental Table 3). Prior studies of lung and ovarian cancers consistently reported that cholesterol levels may affect the efficacy of platinum-based chemotherapeutic agents.50,51 Our findings indicate a new link between genetic variants in BRCA2 and platinum-based chemotherapy response through cholesterol level regulation.