Obesity-Induced Peritoneal Dissemination of Ovarian Cancer and Dominant Recruitment of Macrophages in Ascites

One-fifth of cancer deaths are associated with obesity. Because the molecular mechanisms by which obesity affects the progression of ovarian cancer (OC) are poorly understood, we investigated if obesity could promote the progression of OC cells using the postmenopausal ob/ob mouse model and peritoneal dissemination of mouse ID8 OC cells. Compared to lean mice, obese mice had earlier OC occurrence, greater metastasis throughout the peritoneal cavity, a trend toward shorter survival, and higher circulating glucose and proinflammatory chemokine CXCL1 levels. Ascites in obese mice had higher levels of macrophages (Mφ) and chemokines including CCL2, CXCL12, CXCL13, G-CSF and M-CSF. Omental tumor tissues in obese mice had more adipocytes than lean mice. Our data suggest that obesity may accelerate the peritoneal dissemination of OC through higher production of pro-inflammatory chemokines and Mφ recruitment

CXCR2 is a negative regulator of p21 in p53-dependent and independent manner via Akt-mediated Mdm2 in ovarian cancer

Ovarian cancer (OC) has the highest rate of mortality among gynecological malignancy. Chemokine receptor CXCR2 in OC is associated with poor outcomes. However, the mechanisms by which CXCR2 regulates OC proliferation remain poorly understood. We generated CXCR2-positive cells from parental p53 wild-type (WT), mutant and null OC cells, and assessed the roles of CXCR2 on proliferation of OC cells in p53-dependent and independent manner. CXCR2 promoted cell growth rate: p53WT \u3e mutant = null cells. Nutlin-3, a p53 stabilizer, inhibited cell proliferation in p53WT cells, but had little effect in p53-mutant or null cells, indicating p53-dependence of CXCR2-mediated proliferation. CXCR2 decreased p53 protein, a regulator of p21, and downregulated p21 promoter activity only in p53WT cells. The p53 responsive element (RE) of p21 promoter played a critical role in this CXCR2-mediated p21 downregulation. Moreover, CXCR2-positive cells activated more Akt than CXCR2-negative cells followed by enhanced murine double minute (Mdm2). Silencing Mdm2 or Akt1 upregulated p21 expression, whereas Akt1 overexpression downregulated p21 at the promoter and protein levels in p53WT cells. Cell cycle analysis revealed that CXCR2 decreased p21 gene in p53-null cells. Interestingly, romidepsin (histone deacetylase inhibitor)-induced p21 upregulation did not involve the p53 RE in the p21 promoter in p53-null cells. Romidepsin decreased the protein levels of Akt1 and Mdm2, leading to induction of p21 in p53-null cells. CXCR2 reduced romidepsin-induced p21 upregulation by activating Akt-induced Mdm2. Taken together, CXCR2 enhances cell proliferation by suppressing p21 through Akt-Mdm2 signaling in p53-dependent and independent manner

NF-κB-Mediated CCL20 Reigns Dominantly in CXCR2-Driven Ovarian Cancer Progression.

Ovarian cancer is an inflammation-associated malignancy with a high mortality rate. CXCR2 expressing ovarian cancers are aggressive with poorer outcomes. We previously demonstrated that CXCR2-driven ovarian cancer progression potentiated NF-κB activation through EGFR-transactivated Akt. Here, we identified the chemokine signature involved in CXCR2-driven ovarian cancer progression using a mouse peritoneal xenograft model for ovarian cancer spreading with CXCR2-negative (SKA) and positive (SKCXCR2) cells generated previously from parental SKOV-3 cells. Compared to SKA bearing mice, SKCXCR2 bearing mice had the following characteristics: 1) shorter survival time, 2) greater tumor spreading in the peritoneal cavity and 3) higher tumor weight in the omentum and pelvic site. Particularly, SKCXCR2-derived tumor tissues induced higher activation of the NF-κB signaling pathway, while having no change in EGFR-activated signaling such as Raf, MEK, Akt, mTOR and Erk compared to SKA-derived tumors. Chemokine PCR array revealed that CCL20 mRNA levels were significantly increased in SKCXCR2-derived tumor tissues. The CCL20 promoter activity was regulated by NF-κB dependent pathways. Interestingly, all three κB-like sites in the CCL20 promoter were involved in regulating CCL20 and the proximal region between -92 and -83 was the most critical κB-like site. In addition, SKCXCR2-derived tumor tissues maintained high CCL20 mRNA expression and induced greater CCL24 and CXCR4 compared to SKCXCR2 cells, indicating the shift of chemokine network during the peritoneal spreading of tumor cells via interaction with other cell types in tumor microenvironment. Furthermore, we compared expression profiling array between human ovarian cancer cell lines and tumor tissues based on GEO datasets. The expression profiles in comparison with cell lines revealed that dominant chemokines expressed in ovarian tumor tissues are likely shifted from CXCL1-3 and 8 to CCL20. Taken together, the progression of ovarian cancer in the peritoneal cavity involves NF-κB-mediated CCL20 as a main chemokine network, which is potentiated by CXCR2 expression

Chemokine Network and Overall Survival in TP53 Wild-Type and Mutant Ovarian Cancer

Ovarian cancer (OC) has the highest mortality rate among gynecological malignancies. Because chemokine network is involved in OC progression, we evaluated associations between chemokine expression and survival in tumor suppressor protein p53 (TP53) wild-type (TP53WT) and mutant (TP53m) OC datasets. TP53 was highly mutated in OC compared to other cancer types. Among OC subtypes, CXCL14 was predominantly expressed in clear cell OC, and CCL15 and CCL20 in mucinous OC. TP53WT endometrioid OC highly expressed CXCL14 compared to TP53m, showing better progression-free survival but no difference in overall survival (OS). TP53m serous OC highly expressed CCL8, CCL20, CXCL10 and CXCL11 compared to TP53WT. CXCL12 and CCL21 were associated with poor OS in TP53WT serous OC. CXCR2 was associated with poor OS in TP53m serous OC, while CXCL9, CCL5, CXCR4, CXCL11, and CXCL13 were associated with better OS. Taken together, specific chemokine signatures may differentially influence OS in TP53WT and TP53m OC

NF-κB signaling is significantly activated in the omental tumor tissues from SKCXCR2-bearing mice.

<p>(A) Comparison of signaling pathways in the omental tumor tissues from mice bearing SKA vs. SKCXCR2 cells. The pEGFR (tyr1068), pB-Raf, pc-Raf, pMEK, pAkt, pErk pmTOR and pIκB indicate phosphorylated EGFR, Raf, MEK, Akt, Erk, mTOR and IκB, respectively. β-Actin was used as a loading control. (B) Densitometric comparison of EGFR, Raf, MEK, Akt, Erk, mTOR and IκB activations in the omental tumor tissues from mice bearing SKA vs. SKCXCR2 cells. All data are shown as means ± SE. * indicates a significant increase (p≤0.05) by Student’s-<i>t</i> test.</p

Profiles of chemokine network in human ovarian cancer cell lines and tumor tissues of the cancer associated fibroblasts.

<p>(A) Expression profiling array of chemokine ligand and receptor of 29 human ovarian cancer cell lines from datasets deposited in the NCBI Gene Expression Omnibus (GEO) database (GSE34615). Red dot indicates dominant chemokines in most of the cell lines used. (B) Chemokine and (C) chemokine receptor network in normal ovarian stroma (NOS), ovarian cancer stroma (OCS), ovarian surface epithelium (OSE) and epithelial ovarian cancer (EOC) from datasets deposited in the NCBI GEO database (GSE34615). The intensity of chemokine network was also analyzed. All values are presented as mean. Blue (NOS), yellow (OCS), green (OSE) and red (EOC) dots and letters in chemokine signature indicate dominant chemokine signatures (p≤0.05), respectively, as analyzed by ANOVA and Tukey’s pairwise comparisons. (D) Proposed shift of dominant chemokines between pre-spreading (<i>in vitro</i>) and post-spreading <i>(in vivo</i>) in ovarian cancer.</p

Shift of chemokine network in the tumor tissues of the omentum after implanted with parental cancer cells.

<p>(A) Chemokine and (B) chemokine receptor network in the tumor tissues of the omentum implanted vs. SKA and SKCXCR2 cell line. Expression levels of chemokine network are the means of 4 tumor tissues and cell lines in duplicate, respectively. *,^# indicate increased and decreased chemokines, respectively. (C) Proposed shift of dominant chemokines between pre-spreading and post-spreading of SKCXCR2 cells in the peritoneal cavity. Red letters indicate dominant chemokines in SKCXCR2 cells compared to SKA cells.</p

CCL20 is significantly increased in the omental tumor tissues from SKCXCR2-bearing mice.

<p>(A) Chemokine and (B) chemokine receptor network in the omental tumor tissues from mice bearing SKA vs. SKCXCR2 cells. After isolating total RNA and choosing the qualified RNAs for array, a human chemokine PCR array was performed. Different colors indicate the average cycle threshold with expressions that ranged from >35 to <25. Expression levels of chemokines were defined as absent (>35), low (30–35) and high (<30) on average threshold cycles. Chemokines with a >2-fold increase and average cycle threshold <30 are recognized as induced chemokines, and in this case represents CCL20 (*). (C) Confirmation of CCL20 and CXCR4 mRNA expression in the omental tumor tissues using qRT-PCR. Fold changes were calculated as a relative value after setting the average of SKA tumor tissues as a control group (1.0). All data value are presented as mean ± SE. * indicates a significant increase (p≤0.05) by Student’s-<i>t</i> test.</p

CCL20 promoter activity is tightly regulated by NF-κB signaling.

<p>(A) DNA sequence of human CCL20 promoter. (B) Increased effect of CXCR2 and blockage of IκB on luciferase activity of CCL20 promoter in parental SKOV-3 cells. Results were normalized to the protein level and expressed as a fold increase compared to empty vector (pA) controls. (C) Confirmation of CCL20 promoter activity and its mutants activity in response to TNF (10 ng/ml) in parental SKOV-3 cells. Site-directed mutants were generated from the CCL20-376/20LUC using primers with mutant κB-like sites (termed m1, m2 and m3 indicates mutation site): -219/-210 mutant κB-like site (CCL20LUCm1), -92/-83 mutant κB-like site (CCL20LUCm2) and -33/-24 mutant κB-like site (CCL20LUCm3). After transfection with CCL20 luciferase vectors overnight, a luciferase assay was performed at post-treatment of TNF (10 ng/ml) for 6 h. Results were normalized to the protein level and expressed as a fold increase compared to non-treated control (C). Cross circles indicate κB site mutants. pA = empty vector transfection; pCXCR2 = CXCR2 vector transfection. *,^# indicate significant (p≤0.05) increase and decrease, respectively, when a Student’s-<i>t</i> test was analyzed.</p

SKCXCR2 cells spread more extensively in the peritoneal cavity of mice as compared to SKA cells.

<p>(A) Survival time in mice bearing SKA vs. SKCXCR2 cells. Red numbers indicate the number of dead animals and blue numbers, the number of animals terminated by the accumulation of ascites. (B) The peritoneal spreading tumor burden in mice bearing SKA vs. SKCXCR2 cells. Closed dot lines indicate tumor tissues, white and yellow arrows show detachment and attachment between the diaphragm and the liver, respectively, and carets indicate tumor spots on the liver. (C) The weight of tumor tissues in the diaphragm, omentum and pelvic site obtained from mice bearing SKA vs. SKCXCR2 cells. Sample size was 5 SKA-bearing mice and 4 SKCXCR2-bearing mice terminated by accumulation of ascites and all data are shown as mean ± SE. * indicates a significant increase (p≤0.05) using Student’s-<i>t</i> test.</p