THE ROLE OF GDF15 IN OVARIAN CANCER

Growth Differentiation Factor 15 (GDF15) is induced in situations such as stress, inflammation, treatment with non-steroidal anti-inflammatory drugs, as well as other therapeutic agents. As a secreted protein, GDF15 is seen as a potential biomarker in several types of cancer as well as in other diseases such as cardiovascular diseases, diabetes, and rheumatoid arthritis. In ovarian cancer, high GDF15 serum levels correspond to poor survival. It has further been shown to be expressed at higher levels in serum in ovarian cancer patients post-chemotherapy than pre-chemotherapy. The overall 5-year survival for ovarian cancer is 46%, as a result of late diagnosis when treatment is mostly ineffective. Following initial treatment, 50-75% of patients will develop chemoresistance. Therefore, there is a great interest in identifying markers and therapeutic targets to improve treatment outcome. In this study, we aimed to determine the role that GDF15 plays in the chemoresponse to cisplatin in ovarian cancer. A microarray study identified GDF15 as being among the most highly induced genes following cisplatin treatment of an ovarian cancer cell line. This observation was further verified both in vitro and in vivo. We also found GDF15 induction by platinum agents to be p53 dependent. In addition, in vivo studies of a mouse orthotopic model revealed that GDF15 knockdown tumors were larger than control tumors. The tumors in which GDF15 had been knocked down were smaller following cisplatin treatment. Furthermore, in vivo tumors formed with A2780 ovarian cancer cells in which GDF15 expression was suppressed, demonstrated a reduced percentage of stromal cells compared to tumors formed with control A2780 cells. The stromal percentage of the GDF15 knockdown tumors did not change following cisplatin treatment unlike the control tumors. This study shows for the first time that GDF15 affects tumor composition. In addition, RPPA and RNA-seq was conducted on the mouse tumors to identify downstream targets of GDF15. In summary, this study showed that induction of GDF15 by platinum agents is p53 dependent. This study further showed the effect GDF15 has in ovarian cancer, specifically the tumor composition. This study suggests that targeting GDF15 could be beneficial for patients with p53 wild type ovarian tumors that are often resistant to standard platinum-taxane chemotherapy. Further studies to identify the GDF15 receptor and downstream pathways are necessary

The Role of GDF15 in Regulating the Canonical Pathways of the Tumor Microenvironment in Wild-Type p53 Ovarian Tumor and Its Response to Chemotherapy

Background: The standard treatment of ovarian cancer is surgery followed by a chemotherapeutic combination consisting of a platinum agent, such as cisplatin and a taxane-like paclitaxel. We previously observed that patients with ovarian cancer wild-type for p53 had a poorer survival rate than did those with p53 mutations. Thus, a better understanding of the molecular changes of epithelial ovarian cancer cells with wild-type p53 in response to treatment with cisplatin could reveal novel mechanisms of chemoresistance. Methods: Gene expression profiling was performed on an ovarian cancer cell line A2780 with wild-type p53 treated with cisplatin. A gene encoding a secretory protein growth differentiation factor 15 (GDF15) was identified to be highly induced by cisplatin treatment in vitro. This was further validated in a panel of wild-type and mutant p53 ovarian cancer cell lines, as well as in mouse orthotopic models. The mouse tumor tissues were further analyzed by histology and RNA-seq. Results: GDF15 was identified as one of the highly induced genes by cisplatin or carboplatin in ovarian cancer cell lines with wild-type p53. The wild-type p53-induced expression of GDF15 and GDF15-confered chemotherapy resistance was further demonstrated in vitro and in vivo. This study also discovered that GDF15-knockdown (GDF15-KD) tumors had less stromal component and had different repertoires of activated and inhibited canonical pathways in the stromal cell and cancer cell components from that of the control tumors after cisplatin treatment. Conclusions: GDF15 expression from the wild-type p53 cancer cells can modulate the canonical pathways in the tumor microenvironment in response to cisplatin, which is a possible mechanism of chemoresistance

PAX2 Expression in Ovarian Cancer

PAX2 is one of nine PAX genes that regulate tissue development and cellular differentiation in embryos. However, the functional role of PAX2 in ovarian cancer is not known. Twenty-six ovarian cancer cell lines with different histology origins were screened for PAX2 expression. Two ovarian cancer cell lines: RMUGL (mucinous) and TOV21G (clear cell), with high PAX2 expression were chosen for further study. Knockdown PAX2 expression in these cell lines was achieved by lentiviral shRNAs targeting the PAX2 gene. PAX2 stable knockdown cells were characterized for cell proliferation, migration, apoptosis, protein profiles, and gene expression profiles. The result indicated that these stable PAX2 knockdown cells had reduced cell proliferation and migration. Microarray analysis indicated that several genes involved in growth inhibition and motility, such as G0S2, GREM1, and WFDC1, were up-regulated in PAX2 knockdown cells. On the other hand, over-expressing PAX2 in PAX2-negative ovarian cell lines suppressed their cell proliferation. In summary, PAX2 could have both oncogenic and tumor suppression functions, which might depend on the genetic content of the ovarian cancer cells. Further investigation of PAX2 in tumor suppression and mortality is warranty

PAX2 Expression in Ovarian Cancer

PAX2 is one of nine PAX genes that regulate tissue development and cellular differentiation in embryos. However, the functional role of PAX2 in ovarian cancer is not known. Twenty-six ovarian cancer cell lines with different histology origins were screened for PAX2 expression. Two ovarian cancer cell lines: RMUGL (mucinous) and TOV21G (clear cell), with high PAX2 expression were chosen for further study. Knockdown PAX2 expression in these cell lines was achieved by lentiviral shRNAs targeting the PAX2 gene. PAX2 stable knockdown cells were characterized for cell proliferation, migration, apoptosis, protein profiles, and gene expression profiles. The result indicated that these stable PAX2 knockdown cells had reduced cell proliferation and migration. Microarray analysis indicated that several genes involved in growth inhibition and motility, such as G0S2, GREM1, and WFDC1, were up-regulated in PAX2 knockdown cells. On the other hand, over-expressing PAX2 in PAX2-negative ovarian cell lines suppressed their cell proliferation. In summary, PAX2 could have both oncogenic and tumor suppression functions, which might depend on the genetic content of the ovarian cancer cells. Further investigation of PAX2 in tumor suppression and mortality is warranty

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas.

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a--a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis--as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC50 values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation

Cell viability of ovarian cancer cell lines treated with Nutlin-3a.

<p>All 15 cell lines were plated at a density of 1 × 10³ cells per well in 96-well plates. After 24h, media was exchanged and cells were treated with incremental concentrations of Nutlin-3a (1 μM, 5 μM, 10 μM, 25 μM, 50 μM, and 70 μM). After 72h of Nutlin-3a treatment, cell viability was measured by WST assay and compared to untreated control.</p

Protein expression of <i>TP53</i> and p21 of ovarian cancer cell lines after treated with Nutlin-3a for 24, 48 and 72 hours at their corresponding IC50 as indicated in Table 1.

<p>C, untreated control; *, cancer cell lines carrying <i>TP53</i> mutation. Het, heterozygous TP53 mutation; hom, homozygous <i>TP53</i> mutation.</p

List of genes significantly up-regulated in Nutlin-3a resistant cancer cell lines with wild-type <i>TP53</i>.

<p>List of genes significantly up-regulated in Nutlin-3a resistant cancer cell lines with wild-type <i>TP53</i>.</p

Gene expression of p21, <i>TP53</i>, and MDM2 of ovarian cancer cell lines after treated with Nutlin-3a for 24, 48 and 72 hours at their corresponding IC50 as indicated in Table 1.

<p>(A) <i>TP53</i>. (B) p21. (C) MDM2.</p

<i>TP53</i> mutation status and Nutlin-3a sensitivity of ovarian cancer cell lines.

<p>ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources Cell Bank.</p><p><i>TP53</i> mutation status and Nutlin-3a sensitivity of ovarian cancer cell lines.</p