Qing Sheng

Gli as a Novel Therapeutic Target in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. Current treatment is rarely curative, thus novel meaningful therapies are urgently needed. Inhibition of Hedgehog (Hh) signaling at the cell membrane level in several cancers has shown anti-cancer activity in recent clinical studies. Evidence of Hh-independent Gli activation suggests Gli as a more potent therapeutic target. The current study is aimed to evaluate the potential of Gli as a therapeutic target to treat MPM. The expression profiles of Gli factors and other Hh signaling components were characterized in 46 MPM patient tissue samples by RT-PCR and immunohistochemistry. Cultured cell lines were employed to investigate the requirement of Gli activation in tumor cell growth by inhibiting Gli through siRNA or a novel small molecule Gli inhibitor (Gli-I). A xenograft model was used to evaluate Gli-I in vivo. In addition, a side by side comparison between Gli and Smoothened (Smo) inhibition was conducted in vitro using siRNA and small molecule inhibitors. Our study reported aberrant Gli1 and Gli2 activation in a large majority of tissues. Inhibition of Gli by siRNAs or Gli-I suppressed cell growth dramatically both in vitro and in vivo. Inhibition of Gli exhibited better cytotoxicity than that of Smo by siRNA and small molecule inhibitors vismodegib and cyclopamine. Combination of Gli-I and pemetrexed, as well as Gli-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation in vitro. In summary, Gli activation plays a critical role in MPM. Inhibition of Gli function holds strong potential to become a novel, clinically effective approach to treat MPM.

Inhibition of the Wnt palmitoyltransferase porcupine suppresses cell growth and downregulates the Wnt/β-catenin pathway in gastric cancer

Similarly to the Wnt protein palmitoyltransferase, porcupine (PPN) is essential to the activation of the Wnt/β-catenin signaling pathway. However, little is known about the role of PPN activity in human gastric cancer, one of the most common causes of cancer-related mortality. Real-time quantitative PCR was used to detect the expression levels of PPN in paired gastric cancer tissues. Cell proliferation, migration and invasion assays were performed following treatment using a newly developed small molecule PPN inhibitor (inhibitors of Wnt production, IWP-2) in the gastric cancer MKN28 cell line. Expression of downstream target genes and transcriptional activity of the Wnt/β-catenin signaling pathway were examined following IWP-2 treatment in MKN28. We identified that PPN was overexpressed in human gastric cancer tissue samples and cell lines. Following treatment of the gastric cancer cell line MKN28 with IWP-2, we detected that IWP-2 decreased MKN28 cell proliferation, migration and invasion, and elevated caspase 3/7 activity. Further analysis demonstrated that IWP-2 downregulated the transcriptional activity of the Wnt/β-catenin signaling pathway and downregulated the expression levels of downstream Wnt/β-catenin target genes in MKN28 cells. As current Wnt pathway-targeting strategies used for anticancer therapy have mainly focused on Wnt-receiving cells, our data shed light on the potential use of Wnt palmitoyltransferase PPN inhibitors to abrogate Wnt production in Wnt-producing cells, thus providing a potential therapeutic option for gastric cancer.

Co-targeting of FAK and MDM2 triggers additive anti-proliferative effects in mesothelioma via a coordinated reactivation of p53

Background:Improved mesothelioma patient survival will require development of novel and more effective pharmacological interventions. TP53 genomic mutations are uncommon in mesothelioma, and recent data indicate that p53 remains functional, and therefore is a potential therapeutic target in these cancers. In addition, the tumour suppressor NF2 is inactivated by genomic mechanisms in more than 80% of mesothelioma, causing upregulation of FAK activity. Because FAK is a negative regulator of p53, NF2 regulation of FAK–p53–MDM2 signalling loops were evaluated.Methods:Interactions of FAK–p53 or NF2–FAK were evaluated by phosphotyrosine-p53 immunoaffinity purification and tandem mass spectrometry, and p53, FAK, and NF2 immunoprecipitations. Activation and/or expression of FAK, p53, and NF2 were also evaluated in mesotheliomas. Effects of combination MDM2 and FAK inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, expression of cell cycle checkpoints, and cell cycle alterations.Results:We observed constitutive activation of FAK, a known negative regulator of p53, in each of 10 mesothelioma cell lines and each of nine mesothelioma surgical specimens, and FAK was associated with p53 in five of five mesothelioma cell lines. In four mesotheliomas with wild-type p53, FAK silencing by RNAi induced expression and phosphorylation of p53. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared with four mesothelioma cell lines with nonmutant p53. Additive effects were obtained through a coordinated reactivation of p53, by FAK knockdown/inhibition and MDM2 inhibition, as demonstrated by immunoblots, cell viability, and cell-cycle analyses, showing increased p53 expression, apoptosis, anti-proliferative effects, and cell-cycle arrest, as compared with either intervention alone. Our results also indicate that NF2 regulates the interaction of FAK–p53 and MDM2–p53.Conclusions:These findings highlight novel therapeutic opportunities in mesothelioma.

Adenoviral delivery of the EMX2 gene suppresses growth in human gastric cancer.

Background EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration. Results In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector. Conclusion Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer.

Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107-17. ©2017 AACR.

Abstract 3537: Synergistic effect of a Hedgehog pathway inhibitor in combination with erlotinib on multiple carcinoma cell lines

The aberrant activation of Hedgehog(Hh)/Gli signaling has been implicated in a broad spectrum of malignant tumors. Targeted inhibition at the cell membrane of Hh pathway, for example with Smoothened(Smo) and Hh inhibitors, has shown promising preclinical and clinical anti-cancer activity. However, a growing body of evidence has revealed additional mechanisms in the activation of the Gli pathway that are independent of Hh/Smo regulation. These mechanisms are stimulated by the cross-talk between downstream components of the Hh/Smo and other oncogenic signaling pathways, such as the Epidermal Growth Factor Receptor (EGFR) pathway. Based on this, we hypothesized that the simultaneous inhibition of the EGFR pathway and Gli pathway would result in synergistic anti-tumor effects. To test this hypothesis, we used erlotinib, an EGFR tyrosine kinase inhibitor (TKI), in combination an Hh pathway inhibitor on various lung and mesothelioma cancer cell lines. It has been demonstrated that erlotinib and other TKIs are active in lung cancers with EGFR gene mutations, while not as effective in those with wild type EGFR. However in our studies, the application of the Hh pathway inhibitor sensitized EGFR wild type cells to erlotinib treatment with more than a 10-fold decrease of IC50 in multiple lung cell lines. In addition, the dual treatment resulted in promising synergism in both mesothelioma and lung cancer cell lines with a combination index of less than 0.7. To study the mechanism of crosstalk between the two pathways, we have examined the activities of EGFR, Hh and Wnt pathways in single and dual treatments. Based on our results, we believe that dual inhibition of Hh/Gli and EGFR pathways may be a promising targeted therapy for lung cancers and mesothelioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2011-3537

Comparative study on the composition of free amino acids and derivatives in the two botanical origins of an edible Chinese herb "Xiebai", i.e., Allium chinense G. Don and Allium macrostemon Bunge species.

Xiebai is an edible Chinese herb with various health and therapeutic benefits. To evaluate its nutritional and health values, the free amino acids and derivatives of its two botanical origins (i.e., Allium chinense G. Don and Allium macrostemon Bunge) were isolated using a solvent extraction method and analyzed using automatic amino acid analysis and ultra-performance liquid chromatography-quadrupole-time of flight (UPLC-Q-TOF) mass spectrometry. Our data show that both plants contain abundant free amino acids, and the amount of total free amino acids in A. chinense G. Don is higher than that in A. macrostemon Bunge. The free amino acid compositions in the two plants are qualitatively similar, including nineteen proteinogenic and four non-proteinogenic amino acids. The identified proteinogenic amino acids include eight essential amino acids and five semi-essential amino acids. The sum of essential and semi-essential amino acids accounts for 64.9% and 69.7% of the total free amino acids of the two plants, respectively. The principal amino acids of both plants, from highest concentration to lowest concentration, are arginine, glutamine, glutamic acid, asparagine and serine. A. chinense G. Don is also rich in citrulline and lysine. In addition, two amino acid derivatives were identified from the two plants, i.e., the proline analog N‑methyl‑proline and the dipeptide H-Glu-Tyr-OH. For the first time, the presence of N‑methyl‑proline in the plants of the Allium genus and the presence of H-Glu-Tyr-OH in unprocessed food sources are reported. The influences of the identified substances on the flavor, nutrition and health values of Xiebai are discussed.

Abstract 5136: HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status

Liposarcoma (LPS) is the most common sarcoma of humans. There are no systemic therapeutic regimens known to improve survival when complete surgical resection is not feasible, underscoring the need for an improved molecular understanding of LPS to stimulate the development of effective targeted therapies. The MDM2-p53 pathway plays a prominent role in WDLPS pathogenesis, with the vast majority of human tumors harboring either MDM2 amplifications or p53 mutations. Nutlin-3, a small-molecular antagonist of MDM2, inhibits cell proliferation via blocking the interaction of MDM2-p53 in LPS with wild-type p53. We wonder whether MDM2 overexpression due to amplification or/and mutant p53 still plays a crucial oncogenic role in LPS containing p53 mutation. Herein, we developed isogenic liposarcoma lines in which the parental forms were MDM2 amplification and p53 wild-type, whereas sublines had mutation p53 expression, showed Nutlin-3 resistance. mRNA sequencing and immunoblotting showed that MDM2 and CDK4 were overexpression in the parental lines and sublines, whereas MDM2 and CDK4 expression was low in mesothelioma and GIST cell lines. HDAC inhibitor (HDACi, SAHA and LBH589) treatment resulted in the dephosphorylation and degradation of MDM2 and p53, but little affected CDK4 and JUN expression, irrespective of p53 mutational status in LPS with MDM2 amplification, and in a mesothelioma cell line JMN1B with p53 mutation, but not two mesothelioma lines containing normal MDM2 level and wild-type p53. Our findings indicate that regulation of wild-type 53 degradation by HDACi is MDM2 amplification-dependent. HDAC inhibition by SAHA and LBH589 had a substantially effect on LPS and mesothelioma proliferation and survival associated with upregulation of the PTEN and p21, inhibition of cell proliferation marker cyclin A and PCNA expression, induction of G1 or G2 phase arrest; induction of apoptosis showing an increase of caspase 3/7 activity and expression, PARP cleavage, and the sub-G1 apoptotic population. Moreover, we characterize biological functions of MDM2-p53 axis in nutlin-3-sensitive and nutlin-3-resistant LPS, showing MDM2 knockdown in the four LPS lines and p53 knockdown in the three mutant-p53 LPS lines resulted in anti-proliferative effects. Additive effects were obtained through a coordinated attack on MDM2 and p53, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that MDM2 and p53 knockdown, in LPS cell lines containing the p53 mutation, induced greater cell apoptosis and anti-proliferative effects, compared to either intervention alone, which is comparable to the effects seen after HDAC inhibition by SAHA and LBH589. These compelling pro-apoptotic and anti-proliferative responses indicate that HDAC inhibition warrants clinical evaluation as a novel therapeutic strategy in LPS, including nutlin-3 resistant sublines with the p53 mutation. Note: This abstract was not presented at the meeting. Citation Format: Wen-bin Ou, Hailong Li, Li Liu, Shengmei Zhou, Ye Kuang, Ziqin Yan, Zhe Jiang, Si Shi, Fanguo Meng, Qing Sheng, Haimeng Zhou, Jonathan A. Fletcher. HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5136. doi:10.1158/1538-7445.AM2014-5136

Abstract B212: Inhibition of Gli transcriptional factors sensitizes multiple carcinoma cell lines to erlotinib treatment.

The aberrant activation of Hedgehog/Gli transcriptional factor has been implicated with a broad spectrum of lethal tumors. Targeted inhibition at cell membrane level of the Hh pathway, i.e. Smo and Hh inhibitors, has shown promising anti-tumor activity in both preclinical and clinical trials. A growing body of evidence has revealed additional mechanisms of Gli activation which are independent of Hh/SMO regulation and are stimulated by the crosstalk between components downstream of Hh/Smo and several other oncogenic signaling pathways, such as the Epidermal Growth Factor Receptor (EGFR) pathways. We hypothesized that the simultaneous inhibition of the EGFR pathway and Gli function could abrogate the interaction and result in synergistic anti-tumor effects. In order to test this hypothesis, we have undertaken studies in multiple lung cancer and mesothelioma cell lines. Effects of combination treatments with EGFR tyrosine kinase inhibitor erlotinib and an Hh pathway inhibitor were examined by using MTS assay. In our studies, the application of the Hh pathway inhibitor sensitized lung cancer cells to erlotinib treatment with a higher than 10 fold decrease of IC50. We also used the CalcuSyn software to calculate Combination Index (CI), in order to evaluate the effects of combination treatments (CI 1 indicates Antagonisim). In many lung and mesothelioma cell lines, the dual treatment of the Hh inhibitor and erlotinib showed synergistic effects with a Combination Index (CI) lower than 0.7. To elucidate the mechanism of the crosstalk between the two pathways, we have examined the activities of EGFR and Hh pathways upon single and dual treatments. Based on our studies, we speculate that dual inhibition of Hh/Gli and EGFR pathways may become a promising targeted therapy for lung cancers and mesothelioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B212.

Abstract B183: Nkd2 expression and survival in lung adenocarcinoma.

Objectives: Lung cancer is the leading cause of cancer mortality in the United States and worldwide, and Wnt signaling pathway abnormalities have been implicated in its development. We planned to study the expression of Naked cuticle 2 (Nkd2), a Wnt signaling pathway antagonist, in lung adenocarcinoma tissue specimens. Methods: Two hundred patients with lung adenocarcinoma underwent surgical resection. Tumor and adjacent normal specimens were collected at the time of the procedure and stored in liquid nitrogen until analysis. Clinical data, including patient age, sex, vital status, smoking status, as well as tumor histology and stage, were obtained by review of electronic medical records. RNA was extracted using the Qiagen RNeasy Mini Kit, and cDNA was synthesized using the Bio-Rad iScript Kit. Nkd2 expression was measured using a microarray in 110 patients and quantitative polymerase chain reaction (qPCR) in 90 patients. We looked for differences in Nkd2 expression between patients of different sex, histologic subtype, or vital status; and among patients with different smoking status or stage. We described the distribution of Nkd2 expression in both the microarray and qPCR cohorts. We analyzed the relation between Nkd2 expression and survival using the Kaplan-Meier method and a Cox proportional hazards model. Results: There was no difference in Nkd2 expression between patients of different sex, histologic subtype, or vital status; nor among patients with different smoking status or stage. Nkd2 expression in the tumor was upregulated in the microarray cohort, but downregulated in the qpCR cohort, compared to the adjacent normal. In the microarray cohort, Nkd2 expression was not significantly related to survival (p=0.71). In the qPCR cohort, after adjusting for age, sex, smoking status, bronchoalveolar carcinoma status, and stage, lower Nkd2 expression was significantly associated with longer survival (p=0.03). Stage was the only other significant predictor of survival (Wald test, p = 0.0002). Conclusion: In one cohort in our study, lower Nkd2 expression was significantly associated with longer survival. It is unclear why lower expression of Wnt signaling pathway antagonist would be associated with longer survival, when higher Wnt signaling has been associated with carcinogenesis. One hypothesis is that there is a negative feedback mechanism, in which upregulation of Wnt signaling leads to upregulation of Nkd2. Molecular markers have a growing role in cancer research, and further studies on Nkd2 may lead to new diagnostic and therapeutic applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B183.