Gene expression profile analysis identifies metastasis and chemoresistance-associated genes in epithelial ovarian carcinoma cells

Ovarian cancer has the highest mortality rate of all gynecologic cancers, and 75 % of patients diagnosed with ovarian cancer are already at an advanced stage. Chemotherapy is important in treating and preventing the recurrence of ovarian cancer; however, resistance is an obstacle to overcome and finding new treatment strategies has become increasingly valuable. High-throughput technologies for assaying gene expression, such as high-density oligonucleotide and cDNA microarrays, may offer the potential to identify clinically relevant genes highly differentially expressed between different cell lines. Thus, this study showed the first communication of an investigation that involved the genome-wide examination of differences in gene expression between ovarian cancer cell lines and their sub-lines with enhanced metastasis and chemotherapic resistance. We identified 49 genes that were expressed differentially between Group A, B, C and Group 1, 2, 3, and the average change in expression level between the two groups was at least twofold. The known functions of some of these genes can provide insights with the highly metastasis and chemotherapic resistance for ovarian cancer, although others are still useful for a further research.

GCET2 is found to be the most up-regulated genes in the more enhanced metastasis and chemoresistance cell group, and it is also known as human germinal center associated lymphoma (HGAL) gene, is specifically expressed in germinal center B-lymphocytes and germinal center-derived B cell lymphomas [12], but its function is largely unknown. The GCET2 gene is located on chromosome 3q13 and encodes a 178-amino acid (aa) protein with 51 % identity and 62 % similarity to the murine M17 protein [13]. GCET2 is a cytoplasmatic protein that may also associate with cell membrane. GCET2 expression is associated with improved survival in diffuse large B cell lymphoma (DLBCL) and classic Hodgkin lymphoma patients [14]. In vitro studies in human lymphocytes demonstrated that HGAL increased the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement [15]. In vitro HGAL enhances BCR signaling by binding and increasing Syk activation, in vivo older HGAL transgenic animals progressively developed polyclonal lymphoid hyperplasia and reactive AA amyloidosis [16], these finding suggests that GCET2 may play a role in humoral immune responses. No articles about expression and function of GCET2 on ovarian tissue have been published until now, and our findings make a possible insight of this gene in the study of ovarian cancer, especially about the aspects of metastasis and chemoresistance.

Cystic fibrosis transmembrane conductance regulator (CFTR, ABC35 or ABCC7) was found among the most up-regulated genes in more metastasis and chemoresistance cell line group, and it participates in the beta-adrenergic-dependent CFTR expression pathway. Loss of function mutations of this gene causes the autosomal recessive lethal disease cystic fibrosis (CF) and congenital bilateral aplasia of the vas deferens. There is an increasing interest in the association of cancer incidence with the genetic variations in the CFTR gene. Large cohort studies in North American and European patients with CF found that there was a marked increase in the risk of malignancies affecting the gastrointestinal tract, even to 17 times higher risk of digestive cancer with most cases arising in the bowel [17]. Meanwhile, mutations and low-penetrance polymorphisms in the CFTR gene have been found in patients with various cancers, including pancreatic cancer [18], breast cancer [19], cervical cancer [20], melanoma [21], prostate cancer [22] and lung cancer [23, 24]. On the other hand, CFTR has been suggested to interact with various cancer-related kinases [25]. It encodes a member of the ATP-binding cassette (ABC) transporter superfamily. ABC proteins transport various molecules across extracellular and intracellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). Meanwhile, CFTR is a member of the MRP subfamily that is involved in multidrug resistance. The encoded protein is a cAMP-activated Cl⁻ channel lining the luminal/apical surfaces of epithelial cells in airway, gut, and exocrine glands, and there is a functional coupling between CFTR and MRP2 that may be mediated by PDZ protein [24]. Taken together, our gene expression profile that show a significant up-regulated result of CFTR in more metastasis and chemoresistant ovarian cancer cell lines are consistent with previous findings, a further research on the mechanism of CFTR on ovarian cancer or selective inhibition of CFTR or its pathway may give a insight in therapeutic effects against metastatic and chemoresistant of ovarian cancer.

RBMX gene, also known as HnRNP G, is a member of heterogeneous nuclear ribonucleoprotein (hnRNP) family and can collaborate with hTra2-beta1 (human transformer-2-beta1) as sequence-specific transacting factors to exert antagonistic effects on alternative splicing which is recognized as a pivotal mechanism in regulation of gene expression and associated to tumorigenesis and metastasis of a wide variety of human cancers [26]. It is proposed that the ratio of hnRNP G/hTra2-beta1 influenced cellular splicing preference [27, 28]. Some researches revealed that hnRNP G-protein showed as tumor suppressor in endometrial carcinoma [27] and oral squamous cancer [29], its activity was elicited by transactivating tumor suppressor Txnip gene [30]. A recent research showed that high frequency of hnRNP G-protein reduction and loss of expression in precancerous and human oral squamous cell carcinoma tissue specimens, suggesting that reduction in hnRNP G may play an important role in the early pathogenesis of oral squamous cell carcinomas [31].

GPRC6A encodes an orphan G-protein-coupled receptor, mediates the non-genomic effects of testosterone and other anabolic steroids in multiple tissue, and it is a potential target for developing antagonists and agonists that would have broad applications in health and disease [32], including cancer. A genome-wide association study on prostate cancer identified GPRC6A was one of the five novel genetic loci associated with prostate cancer in Japanese and Chinese Han population [33, 34], and the same result was verified by a genome-wide testing of putative functional exonic variants in a multiethnic population [35]. GPRC6A is expressed at higher levels in human prostate cancer cells and prostate cancer tissues and small interfering RNA knockdown of GPRC6A attenuates these response in human prostate cancer cell lines [36]. GPRC6A is also coupled to signaling pathways, such as phosphatidylinositol 3-kinase that are known to be deregulated in prostate cancer [32]. On all accounts, nearly all researches about GPRC6A associated with cancer focused on prostate cancer, no investigation about this gene on ovarian cancer has been published.

FOXP1, as one of the down-regulated genes, drew our attention for a further research. FOXP1 is a FOX family member consisting of the winged-helix DNA-binding domain and the N-terminal transcriptional repression domain, and it is widely expressed and plays a key role in the development of various human tissues [37, 38]. FOXP1 represses its target genes by forming homodimers or heterodimers with FOXP2 and FOXP4 [39], it has been suggested to be both a tumor suppressor candidate and potential oncogene, because of its differential expression levels in distinctive types of tumors, including B cell lymphomas [40], breast cancer [41, 42], endometrial cancer [43], prostate cancer [44], non-small cell lung cancer [38] and renal cell carcinoma [45], the loss of FOXP1 in breast cancer has been associated with shorter survival times [42]. Until now, no article about FOXP1 expression in ovarian cancer has been published, and we made the first investigation of FOXP1 protein expression in ovarian tissue and found that nuclear staining of FOXP1 decreased as the metastasis increased, a significant decrease in FOXP1 expression in the resistance group, nuclear FOXP1 expression were independent risk factors strongly correlated with prognosis of ovarian cancer, above all, FOXP1 may serve as a good marker for late stage ovarian cancer and chemoresistance EOC patients, high expression of FOXP1 in nucleus is associated with improved survival in patients with ovarian cancer.

There are some pseudogenes which shows significant expression difference in enhanced metastasis and chemoresistant ovarian cancer cell lines, in which BC031676 and BC113708 are up-regulated, and RPL28P1, RPL23A, RPL13AP3, LOC341412, LOC641784, LOC391560, RPS16P9, LOC732186, RPL13AP23, RPLP1P7, RPL31P10, LOC648361 are down-regulated, and most of them are ribosomal protein pseudogenes. Pseudogenes are DNA sequences similar to genes encoding functional proteins but are presumed to be nonfunctional due to mutations and truncation by premature stop codons [46]. Ribosomal protein (RP) pseudogenes constitute the largest family of pseudogenes (approximately 2000 RP processed pseudogenes), and they are constitutively expressed at reasonably stable levels and are very highly conserved [47]. Although pseudogenes have long been considered as nonfunctional genomic sequences, during recent two decades, especially with the broad applications of next-generation sequencing technologies, emerging evidences have confirmed that some pseudogenes have acquired diverse functions in regulating development and diseases, especially in cancers [48]. Some pseudogenes are specifically expressed in certain cancers or diseases. It has been shown that the pseudogene of PTEN, PTENP1, was selectively lost in some human cancer cells, resulting in decreased expression of PTEN and abnormal proliferation of cancer cells [49]. The expression of MYLKP1, a duplicated pseudogene of MYLK, can decrease the stability of MYLK mRNA at the posttranscriptional level and stimulate cell proliferation [50]. Recently, a study provided a systematic approach to analyze expressed pseudogenes, enabling comparisons of cancer versus benign tissues in multiple solid tumors, which overcome the limitations of previous analyses of pseudogene expression. They observed 218 pseudogenes expressed only in cancer samples, of which 178 were observed in multiple cancers, and 40 were found to have highly specific expression in a single cancer type only, finally they described ATP8A2-J and CXADR-J pseudogenes preferentially associated with distinct subsets of breast cancer and prostate cancer patients, respectively [51]. Besides cancers, pseudogenes also involve in the development of other diseases, such as HMGA1-p [52]. Although the regulatory functions of pseudogenes seem to be striking, the functional studies of pseudogenes are still in its early stage. The pseudogenes in our study should not be useless, their functions and relationships with ovarian cancer, especially the enhanced metastasis and chemoresistance should be investigated in near further.

In conclusion, this study has identified potential DEGs responsible for enhanced metastasis and chemoresistance in ovarian cancer cell lines. Among the 49 DEGs, 14 genes were up-regulated and 35 genes were down-regulated. Prospective investigations using a combination of genomic and proteomic approaches are required to validate the functionality of these targets identified.