Wei Hu’s research while affiliated with University of Texas MD Anderson Cancer Center and other places

EP-100 is a synthetic lytic peptide that specifically targets the gonadotropin-releasing hormone receptor on cancer cells. To extend the utility of EP-100, we aimed to identify effective combination therapies with EP-100 for ovarian cancer and explore potential mechanisms of this combination. A series of in vitro (MTT assay, immunoblot analysis, reverse-phase protein array, comet assay, and immunofluorescence staining) and in vivo experiments were carried out to determine the biological effects of EP-100 alone and in combination with standard-of-care drugs. EP-100 decreased the viability of ovarian cancer cells and reduced tumor growth in orthotopic mouse models. Of five standard drugs tested (cisplatin, paclitaxel, doxorubicin, topotecan, and olaparib), we found that the combination of EP-100 and olaparib was synergistic in ovarian cancer cell lines. Further experiments revealed that combined treatment of EP-100 and olaparib significantly increased the number of nuclear foci of phosphorylated histone H2AX. In addition, the extent of DNA damage was significantly increased after treatment with EP-100 and olaparib in comet assay. We performed reverse-phase protein array analyses and identified that the PI3K/AKT pathway was inhibited by EP-100, which we validated with in vitro experiments. In vivo experiment using the HeyA8 mouse model demonstrated that mice treated with EP-100 and olaparib had lower tumor weights (0.06 ± 0.13 g) than those treated with a vehicle (1.19 ± 1.09 g), EP-100 alone (0.62 ± 0.78 g), or olaparib alone (0.50 ± 0.63 g). Our findings indicate that combining EP-100 with olaparib is a promising therapeutic strategy for ovarian cancer.

For mucinous ovarian cancer (MOC), standard platinum-based therapy is largely ineffective. We sought to identify possible mechanisms of oxaliplatin resistance of MOC and develop strategies to overcome this resistance. A kinome-based siRNA library screen was carried out using human MOC cells to identify novel targets to enhance the efficacy of chemotherapy. In vitro and in vivo validations of antitumor effects were performed using mouse MOC models. Specifically, the role of PRKRA/PACT in oxaliplatin resistance was interrogated. We focused on PRKRA, a known activator of PKR kinase, and its encoded protein PACT because it was one of the five most significantly downregulated genes in the siRNA screen. In orthotopic mouse models of MOC, we observed a significant antitumor effect of PRKRA siRNA plus oxaliplatin. In addition, expression of miR-515-3p was regulated by PACT-Dicer interaction, and miR-515-3p increased the sensitivity of MOC to oxaliplatin. Mechanistically, miR-515-3p regulated chemosensitivity, in part, by targeting AXL. The PRKRA/PACT axis represents an important therapeutic target in MOC to enhance sensitivity to oxaliplatin.

Mounting clinical and preclinical evidence supports a key role for sustained adrenergic signaling in the tumor microenvironment as a driver of tumor growth and progression. However, the mechanisms by which adrenergic neurotransmitters are delivered to the tumor microenvironment are not well understood. Here we present evidence for a feed-forward loop whereby adrenergic signaling leads to increased tumoral innervation. In response to catecholamines, tumor cells produced brain-derived neurotrophic factor (BDNF) in an ADRB3/cAMP/Epac/JNK-dependent manner. Elevated BDNF levels in the tumor microenvironment increased innervation by signaling through host neurotrophic receptor tyrosine kinase 2 receptors. In patients with cancer, high tumor nerve counts were significantly associated with increased BDNF and norepinephrine levels and decreased overall survival. Collectively, these data describe a novel pathway for tumor innervation, with resultant biological and clinical implications. Significance: Sustained adrenergic signaling promotes tumor growth and metastasis through BDNF-mediated tumoral innervation. Cancer Res; 78(12); 3233–42. ©2018 AACR.

Thrombocytosis is present in more than 30% of patients with solid malignancies and correlates with worsened patient survival. Tumor cell interaction with various cellular components of the tumor microenvironment including platelets is crucial for tumor growth and metastasis. Although it is known that platelets can infiltrate into tumor tissue, secrete pro-angiogenic and pro-tumorigenic factors and thereby increase tumor growth, the precise molecular interactions between platelets and metastatic cancer cells are not well understood. Here we demonstrate that platelets induce resistance to anoikis in vitro and are critical for metastasis in vivo. We further show that platelets activate RhoA-MYPT1-PP1-mediated YAP1 dephosphorylation and promote its nuclear translocation which induces a pro-survival gene expression signature and inhibits apoptosis. Reduction of YAP1 in cancer cells in vivo protects against thrombocytosis-induced increase in metastasis. Collectively, our results indicate that cancer cells depend on platelets to avoid anoikis and succeed in the metastatic process.

Background and objectives: To examine association of lympho-vascular space invasion (LVSI) with clinico-pathological factors and to evaluate survival of women with low-grade serous ovarian carcinoma containing areas of LVSI. Methods: This is a multicenter retrospective study examining consecutive cases of surgically treated stage I-IV low-grade serous ovarian carcinoma (n = 178). Archived histopathology slides for the ovarian tumors were reviewed, and LVSI was scored as present or absent. LVSI status was correlated to clinico-pathological findings and survival outcome. Results: LVSI was seen in 79 cases (44.4%, 95% confidence interval [CI] 37.1-51.7). LVSI was associated with increased risk of omental metastasis (87.0% vs 64.9%, odds ratio [OR] 3.62, P = 0.001), high pelvic lymph node ratio (median 12.9% vs 0%, P = 0.012), and malignant ascites (49.3% vs 32.6%, OR 2.01, P = 0.035). On multivariable analysis, controlling for age, stage, and cytoreductive status, presence of LVSI in the ovarian tumor remained an independent predictor for decreased progression-free survival (5-year rates 21.0% vs 35.7%, adjusted-hazard ratio 1.57, 95%CI 1.06-2.34, P = 0.026). LVSI was significantly associated with increased risk of recurrence in lymph nodes (OR 2.62, 95%CI 1.08-6.35, P = 0.047). Conclusion: LVSI in the ovarian tumor is associated with adverse clinico-pathological characteristics and decreased progression-free survival in women with low-grade serous ovarian carcinoma.

Purpose: Chronic adrenergic activation has been shown to associate with adverse clinical outcomes in cancer patients, but the underlying mechanisms are not well understood. The focus of the current study was to determine the functional and biological effects of adrenergic pathways on response to chemotherapy in the context of ovarian cancer. Experimental design: Increased DUSP1 production by sympathetic nervous system mediators (e.g., norepinephrine) was analyzed by real-time quantitative RT-PCR and by Western blotting. In vitro chemotherapy-induced cell apoptosis was examined by flow cytometry. For in vivo therapy, a well-characterized model of chronic stress was used. Results: Catecholamines significantly inhibited paclitaxel- and cisplatin-induced apoptosis in ovarian cancer cells. Genomic analyses of cells treated with norepinephrine identified DUSP1 as a potential mediator. DUSP1 overexpression resulted in reduced paclitaxel-induced apoptosis in ovarian cancer cells compared with control; conversely, DUSP1 gene silencing resulted in increased apoptosis compared to control cells. DUSP1 gene silencing in vivo significantly enhanced response to paclitaxel and increased apoptosis. In vitro analyses indicated that norepinephrine-induced DUSP1 gene expression was mediated through ADRB2 activation of cAMP-PLC-PKC-CREB signaling, which inhibits JNK-mediated phosphorylation of c-Jun and protects ovarian cancer cells from apoptosis. Moreover, analysis of The Cancer Genome Atlas data showed that increased DUSP1 expression was associated with decreased overall (P = 0.049) and progression-free (P = 0.0005) survival. Conclusions: These findings provide a new understanding of the mechanisms by which adrenergic pathways can impair response to chemotherapy and have implications for cancer management.

The aim of this article was to review the recent literature on potential therapeutic strategies for overcoming resistance to antivascular endothelial growth factor drugs in ovarian cancer. Although clinical benefits of antivascular endothelial growth factor therapy were observed in ovarian cancer treatment trials, this use yielded only modest improvement in progression-free survival and, with the exception of cediranib, no effect on overall survival. Adaptive resistance and escape from antiangiogenesis therapy is likely a multifactorial process, including induction of hypoxia, vascular modulators, and immune response. New drugs targeting the tumor vasculature or other components of the surrounding microenvironment have shown promising results. When to start and end antiangiogenesis therapy and the choice of optimal treatment combinations remain controversial. Further evaluation of personalized novel angiogenesis-based therapy is warranted. Defining the critical interaction of these agents and pathways and the appropriate predictive markers will become an increasingly important objective for effective treatment.

Background Early detection of ovarian cancer remains a challenge due to widespread metastases and a lack of biomarkers for early-stage disease. This study was conducted to identify relevant biomarkers for both laparoscopic and serum diagnostics in ovarian cancer.Methods Bioinformatics analysis and expression screening in ovarian cancer cell lines were employed. Selected biomarkers were further validated in bio-specimens of diverse cancer types and ovarian cancer subtypes. For non-invasive detection, biomarker proteins were evaluated in serum samples from ovarian cancer patients.ResultsTwo kallikrein (KLK) serine protease family members (KLK6 and KLK7) were found to be significantly overexpressed relative to normal controls in most of the ovarian cancer cell lines examined. Overexpression of KLK6 and KLK7 mRNA was specific to ovarian cancer, in particular to serous and papillary serous subtypes. In situ hybridization and histopathology further confirmed significantly elevated levels of KLK6 and KLK7 mRNA and proteins in tissue epithelium and a lack of expression in neighboring stroma. Lastly, KLK6 and KLK7 protein levels were significantly elevated in serum samples from serous and papillary serous subtypes in the early stages of ovarian cancer, and therefore could potentially decrease the high ¿false negative¿ rates found in the same patients with the common ovarian cancer biomarkers human epididymis protein 4 (HE4) and cancer antigen 125 (CA-125).ConclusionKLK6 and KLK7 mRNA and protein overexpression is directly associated with early-stage ovarian tumors and can be measured in patient tissue and serum samples. Assays based on KLK6 and KLK7 expression may provide specific and sensitive information for early detection of ovarian cancer.

Objective: Members of the Eph/Ephrin family are known to play seminal roles in cancer growth and metastasis in many solid tumors; however, a comprehensive analysis has not been carried out. We analyzed the clinical relevance of all Eph/Ephrin family members in endometrial cancer. Methods: Level 3 data from The Cancer Genome Atlas (TCGA) was utilized to assess the clinical significance of all 14 Eph receptors available in the database (EphA1-8, EphA10, EphB1-4, and EphB6), as well as EphrinA1-A5 and EphrinB1-B3. We examined gene methylation status and copy number changes in these tumors. Clinical information extracted included age, tumor histology, tumor grade, stage, estrogen(ER)/progesterone(PR) receptor status, and overall survival. Results: A total of 320 samples were available for analysis. Increased EphA2, EphA4, EphB1, and EphB2 expression was significantly correlated with poorer overall survival (p=0.03, p=0.04, p=0.01, and p=0.008, respectively) and lack of ER and PR expression (r=-0.34, p=2.25e−6; r-0.24, p=0.0007; r=-0.22, p=0.002; r=-0.46, p=2.38e−11, respectively). Compared to endometrioid histology, serous histology was associated with increased expression of EphA2 (p<0.0001), EphA4 (p<0.0002), EphB1 (p<0.0001), EphB2 (p<0.0001), and EphrinA3 (p=0.04). Grade 3 tumor samples had increased expression of EphA2 (p<0.03), EphA4 (p<0.006), EphB1 (p<0.0001), EphB2 (p<0.0001), and EphrinA3 (p=0.04) compared to lower grade samples. EphA2, EphB1, and EphB2 expression was higher in samples from patients with advanced stage (FIGO Stage IIIA-IVB) compared to those with early stage disease (p=0.008, p=0.0183, and p<0.001, respectively). Patients age > 63 years had greater tumoral expression of EphA4 (p<0.0001), EphB2 (p=0.001), and EphrinA3 (p=0.03). Increased copy number correlated with EphB2 (r=0.41, p=0.001) and EphrinA3 (r=0.23, p=0.02) expression, but was not correlated with other Eph or Ephrin gene expression. Gene methylation inversely correlated with EphA2, EphA4, EphB1, EphB2, EphrinA3, and EphrinA5 receptor expression (r<-0.22, p<0.0001; r<-0.26, p<0.0002; r<-0.23, p<0.0006; r<-0.21, p<0.003; r<-0.2, p<0.01; r<-0.2, p<0.01, respectively). In patients with advanced stage expression of EphrinA3 and EphrinA5 was associated with poorer OS (p=0.002 and p=0.012). In a multivariate analysis, stage and Eph expression were the strongest independent predictors of poor survival, of which, EphA4 was the most significant (p=0.0024). In further support of the significance of EphA4, increased expression of its ligands EphrinA3 and EprhinA5 correlated with poorer OS. Conclusions: Concordant with emerging roles for the Eph receptor and Eprhins in driving malignant biology, increased EphA2, EphA4, EphB1, and EphB2 expression, as well as EphrinA3 and EphrinA5 expression, is predictive of poor patient outcome and constitutes attractive targets for biological therapies. Citation Format: Heather J. Dalton, Cristina Ivan, Chad V. Pecot, Rajesha Rupamaiole, Behrouz Zand, Justin Bottsford-Miller, Wei Hu, Alpa M. Nick, Robert L. Coleman, Anil K. Sood. An integrated analysis of the eph/ephrin family: implications in endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4034. doi:10.1158/1538-7445.AM2013-4034

Objective: To identify genomic predictors of overall survival of women with high grade serous ovarian cancer based on best separation overall survival Kaplan-Meier curves Methods: Comprehensive data analysis of the TCGA dataset of high grade serous epithelial ovarian cancer was carried out with a “training” (n= 375) and “validation” (n= 188) data sets. The R-code was written specifically to identify all genes with high expression that gave the greatest predictive potential for patient survival. The top ten cases with lowest sums of the log rank test p-values obtained for training and validation sets were chosen to be included with Kaplan-Meier survival curves for analysis. Results: The ten genes with high expression that had the smallest p-values in their respective order were: SLC6A1, LIPK, EHBP1, SUSD5, PEX3, SLC22A3, RABGEF1, PPM2C, KIAA1219, and GALNT10. The range of p-value sum ranged from 1.77E-3 to 2.09E-3. The lowest p-value was for SLC6A1, which encodes for GABA Transporter-1 that removes GABA from extracellular to intracellular space. Interestingly, four of the 10 genes predictive for poor outcome are directly involved in cell metabolism: LIPK, PPM2C, PEX3, and GALNT10. The median OS of the high gene expression group ranged from 38 to 48 months. The median OS of the low gene expression group ranged from 55-70 months. The median OS difference of high gene vs. low gene expression groups ranged from 15-25 months. EHBP1 gene, involved in endocytic trafficking of transferrin into endosomes and GLUT4 into adipocytes, had the greatest difference in median OS between high and low expressions at 25 months (p= 2.16E-3). Finally, low expression of PPM2C compared to its high expression was significantly associated with > 5 year survival (10.1% vs. 1.1%, p=0.001) and no recurrence in both training and validation sets (12.5% vs. 2.2%, p=0.04). PPM2C encodes for pyruvate dehydrogenase-phosphatase 1 that catalyzes reactivation of the alpha subunit of the E1 component of the pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex in the active form transforms pyruvate from glycolysis to acetyl-CoA in the mitochondria. To evaluate whether similar findings are noted in other cancers, we also examined the TCGA data for breast, lung, colon cancer, and glioblastoma. PPM2C is amplified by 7% in invasive breast cancer, and leads to an overall worse survival (68.9 months vs. 129.5 months, p = 0.037). Conclusion: Genes involved in cell metabolism, neurotransmitter functioning, and endocytic trafficking are highly predictive of outcome in high grade serous ovarian cancer. High PPM2C expression and gene amplification leads to worsening overall survival in ovarian and invasive breast cancer, respectively. These pathways likely reflect novel and important therapeutic targets. Citation Format: Behrouz Zand, Cristina Ivan, Chad V. Pecot, Rajesha Rupaimoole, Heather J. Dalton, Justin Bottsford-Miller, Wei Hu, Alpa M. Nick, Anil K. Sood. Ten degrees of separation: novel genes affecting outcome in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2013-4024