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Genomic MET amplification and exon 14 skipping are currently clinically recognized biomarkers for stratifying subsets of non-small cell lung cancer (NSCLC) patients according to the predicted response to c-Met inhibitors (c-Metis), yet the overall clinical benefit of this strategy is quite limited. Notably, c-Met protein overexpression, which occurs in approximately 20–25% of NSCLC patients, has not yet been clearly defined as a clinically useful biomarker. An optimized strategy for accurately classifying patients with c-Met overexpression for decision-making regarding c-Meti treatment is lacking. Herein, we found that SYK regulates the plasticity of cells in an epithelial state and is associated with their sensitivity to c-Metis both in vitro and in vivo in PDX models with c-Met overexpression regardless of MET gene status. Furthermore, TGF-β1 treatment resulted in SYK transcriptional downregulation, increased Sp1-mediated transcription of FRA1, and restored the mesenchymal state, which conferred resistance to c-Metis. Clinically, a subpopulation of NSCLC patients with c-Met overexpression coupled with SYK overexpression exhibited a high response rate of 73.3% and longer progression-free survival with c-Meti treatment than other patients. SYK negativity coupled with TGF-β1 positivity conferred de novo and acquired resistance. In summary, SYK regulates cell plasticity toward a therapy-sensitive epithelial cell state. Furthermore, our findings showed that SYK overexpression can aid in precisely stratifying NSCLC patients with c-Met overexpression regardless of MET alterations and expand the population predicted to benefit from c-Met-targeted therapy.

Lactate accumulation in the tumor microenvironment was shown to be closely related to tumor growth and immune escape, and suppression of lactate production by inhibiting lactate dehydrogenase A (LDHA) has been pursued as a potential novel antitumor strategy. However, only a few potent LDHA inhibitors have been developed and most of them did not show potent antitumor effects in vivo. To this end, we designed new LDHA inhibitors and obtained a novel potent LDHA inhibitor, ML-05. ML-05 inhibited cellular lactate production and tumor cell proliferation, which was associated with inhibition of ATP production and induction of reactive oxygen species and G

Background The interaction between tumor cells and their immunosuppressive microenvironment promotes tumor progression and drug resistance. Thus, simultaneously targeting tumor cells and stromal cells is expected to have synergistic antitumor effects. Herein, we present for the first time a preclinical antitumor investigation of 3D185, a novel dual inhibitor targeting FGFRs, which are oncogenic drivers, and CSF-1R, which is the major survival factor for protumor macrophages. Methods The antitumor characteristics of 3D185 were assessed by a range of assays, including kinase profiling, cell viability, cell migration, immunoblotting, CD8+ T cell suppression, and in vivo antitumor efficacy, followed by flow cytometric and immunohistochemical analyses of tumor-infiltrating immune cells and endothelial cells in nude mice and immune-competent mice. Results 3D185 significantly inhibited the kinase activity of FGFR1/2/3 and CSF-1R, with equal potency and high selectivity over other kinases. 3D185 suppressed FGFR signaling and tumor cell growth in FGFR-driven models both in vitro and in vivo. In addition, 3D185 could inhibit the survival and M2-like polarization of macrophages, reversing the immunosuppressive effect of macrophages on CD8+ T cells as well as CSF1-differentiated macrophage induced-FGFR3-aberrant cancer cell migration. Furthermore, 3D185 inhibited tumor growth via remodeling the tumor microenvironment in TAM-dominated tumor models. Conclusions 3D185 is a promising antitumor candidate drug that simultaneously targets tumor cells and their immunosuppressive microenvironment and has therapeutic potential due to synergistic effects. Our study provides a solid foundation for the investigation of 3D185 in cancer patients, particularly in patients with aberrant FGFR and abundant macrophages, who respond poorly to classic pan-FGFRi treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1357-y) contains supplementary material, which is available to authorized users.

Purpose: Tumor heterogeneity may represent a barrier to preoperative genomic characterization by needle biopsy in clear cell renal cell carcinoma (ccRCC). The extent of heterogeneity in small renal tumors remains unknown. Therefore, we set out to evaluate heterogeneity in resected large and small renal tumors. Experimental Design: We conducted a study from 2013 to 2016 that evaluated 47 consecutive ccRCC tumors resected during radical or partial nephrectomy. Cases were designated as small (7 cm) tumors. Each tumor had three regions sampled. Copy-number variation (CNV) was assessed and gene expression analysis was performed to characterize the clear-cell A and B (ccA/ccB) profile and the cell-cycle progression (CCP) score. Genomic heterogeneity between three regions was evaluated using CNV subclonal events, regional expression profiles, and correlation between gene expression. Results: Twenty-three small and 24 large tumors were analyzed. Total CNVs and subclonal CNVs events were less frequent in small tumors (P < 0.001). Significant gene expression heterogeneity was observed for both CCP scores and ccA/ccB classifications. Larger tumors had more variance in CCP scores (P = 0.026). The distribution of ccA/ccB differed between small and large tumors with mixed ccA/ccB tumors occurring more frequently in the larger tumors (P = 0.024). Analysis of five mixed tumors (with both ccA/ccB regions) demonstrated the more aggressive ccB phenotype had greater CNV events (P = 0.014). Conclusions: Small renal tumors have much less genomic complexity and fewer subclonal events. Pretreatment genomic characterization with single-needle biopsy in small tumors may be useful to assess biologic potential and may influence therapy. Clin Cancer Res; 24(17); 4137–44. ©2018 AACR.

The roles of Ephrin B (EphB) receptors in cancer are relatively unknown as these receptors are associated with complex signaling pathways. A limited number of studies have investigated the association between EphB receptors and prognosis. Using the Kaplan-Meier plotter database, the present study investigated the associations between the mRNA expression levels of five EphB receptors and the outcomes of 3,554 patients with breast cancer who had been followed-up for 20 years. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated to assess the relative risk of survival. The results demonstrated that high mRNA expression levels of EphB2 (HR, 0.74; 95% CI, 0.66–0.84; P=2.1×10-6), EphB4 (HR, 0.82; 95% CI, 0.72–0.93; P=0.0023) and EphB6 (HR, 0.69; 95% CI, 0.61–0.78; P=3×10-9) were significantly associated with improved survival, while a high mRNA expression level of EphB3 (HR, 1.14; 95% CI, 1.01–1.28; P=0.029) was associated with worse survival for patients with breast cancer. High expression levels of all EphB receptors, including EphB1 (HR, 1.4; 95% CI, 1.02–1.94; P=0.039), EphB2 (HR, 1.34; 95% CI, 1.07–1.67; P=0.011), EphB3 (HR, 1.39; 95% CI, 1.11–1.73, P=0.0038), EphB4 (HR, 1.33; 95% CI, 1.06–1.67; P=0.013) and EphB6 (HR, 1.32; 95% CI, 1.05–1.65; P=0.016), were associated with an increased risk of mortality in patients with lymph-node-positive breast cancer. High mRNA expression levels of EphB1 were not associated with survival for all patients with breast cancer (HR, 0.85; 95% CI, 0.72–1.01; P=0.058). The results of the present suggested that EphB receptors may be useful as prognostic biomarkers of breast cancer.

Ceramides have emerged as potential therapeutic option with novel mechanism to affect the proliferation, differentiation, senescence, and apoptosis of cancer cells through regulation of multiple signal transduction. Aiming at the improvement of the apoptosis activity and pharmacokinetic profiles of ceramides, a novel series of ceramide analogs were developed through structure simplification and conformation restriction. Among them, compound 12 bearing an alkoxyl naphthyl motif, with favorable rat pharmacokinetic properties, showed better anti-proliferative activity against various colon cancer cells (IC50 ∼20 μM) than other ceramide analogues, as well as the synergistic effect combined with AKT inhibitor MK2206. Additionally, we demonstrated that this combination therapy promoted caspase 3-dependent apoptotic pathway and intensified cell cycle arrest in the G0/G1 phase. Furthermore, the combination of compound 12 and MK2206 displayed synergistic anti-tumor effect in vivo.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor with high metastatic potential. However, no good biomarker has been identified to refine which subtype is of high metastatic potential to make decisions regarding the elective and therapeutic management of lymphatic metastases. In this study, we investigated the role of the metabolic enzyme phosphoglycerate mutase 1 (PGAM1) in OSCC. PGAM1 expression was examined in tissue samples of 122 OSCC patients using immunohistochemistry, and the correlation between clinicopathological expression and PGAM1 expression was determined. Survival curves were generated using the Kaplan-Meier method, and multivariate analysis was performed by the Cox proportional hazards model. Moreover, PGAM1 was knocked down in the OSCC cell lines Cal27 and HN12, followed by determination of the change in cell migration and signaling pathways. PGAM1 expression is correlated with age, lymphatic metastasis and tumor recurrence and is closely associated with poor overall survival (OS) and disease-free survival (DFS). Intriguingly, PGAM1 is an independent risk factor for OS and DFS. After knocking down PGAM1 in Cal27 and HN12 cells, cell migration was remarkably decreased along with signaling pathway molecules, such as proto-oncogene c-SRC (SRC), Focal adhesion kinase (FAK) and Paxillin. The effect on cell migration was abolished following pretreatment with an SRC inhibitor. This study suggested that PGAM1 is a poor prognostic biomarker of OSCC and may be used to select patients of high metastatic potential in the clinic, and PGAM1 promotes the migration of OSCC cells is associated with the SRC pathway.

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that coordinates glycolysis and biosynthesis to promote cancer growth via its metabolic activity. Here, we report the discovery of a non-metabolic function of PGAM1 in promoting cancer metastasis. A proteomic study identified α-smooth muscle actin (ACTA2) as a PGAM1-associated protein. PGAM1 modulated actin filaments assembly, cell motility and cancer cell migration via directly interacting with ACTA2, which was independent of its metabolic activity. The enzymatically inactive H186R mutant retained its association with ACTA2, whereas 201-210 amino acids deleted PGAM1 mutant lost the interaction with ACTA2 regardless of intact metabolic activity. Importantly, PGAM1 knockdown decreased metastatic potential of breast cancer cells in vivo and PGAM1 and ACTA2 were jointly associated with the prognosis of breast cancer patients. Together, this study provided the first evidence revealing a non-metabolic function of PGAM1 in promoting cell migration, and gained new insights into the role of PGAM1 in cancer progression.

Purpose: Sphingosine-1-phosphate (S1P) is a pleotropic bioactive lipid mediator involved in the pathogenesis of inflammation and cancer. FTY720 is a “functional antagonist” of S1P by binding and induces the internalization and degradation of S1P receptors. Indeed, FTY720 was shown to have antitumor activity in several tumor models, while whether its immune-suppressive activity promotes or inhibits tumor growth is still unclear. In this study, we explored the antitumor activity of FTY720 in various cancer cells and its underlying mechanisms. Experimental Procedures: We analyzed the antiproliferative activity of FTY720 in nearly 30 human cancer cell lines and 6 mouse cancer cell lines. Human colon cancer cells SW620 and mouse breast cancer cells 4T1, which are sensitive to FTY720, were selected and used for in vivo study. SW620 was subcutaneously inoculated into Balb/c nude mice and 4T1 were in situ inoculated into immune-competent Balb/c mice. Mice was administrated intraperitoneally with low dose (0.5 mg/kg/d) and high dose (5 mg/kg/d) of FTY720. Tumor volume and body weight were measured every 3 days. After 3 weeks, the mice were sacrificed, and the tumor was removed and weighed, and kept for further analysis. The signaling pathway involved in S1P pathway and neovascularization were analyzed. In addition, the pharmacokinetics of FTY720 was also determined in mice. The effect of FTY720 on angiogenesis was also analyzed at cellular level, as well as the gene expression in HUVEC cells. Summary: FTY720 showed potent antiproliferative activity across various cancer cell lines. The IC50 for SW620 and 4T1 was 3.71 μM and 3.38 μM respectively, which were most sensitive. In animal study, both low-dose and high-dose FTY720 dramatically inhibited the tumor growth in SW620 xenograft model, while only high-dose FTY720 inhibited tumor growth in 4T1 xenograft model. Since the serum concentration of FTY720 was much lower than its cytotoxicity at low dose, we thus propose the anti-umor activity of FTY720 may due to its antiangiogenic activity. Moreover, the antitumor activity of FTY720 was much lower for 4T1 as compared to SW620 xenograft model, which possibly is caused by its immune-suppressive activity as it depleted the circulating lymphocytes. Besides, FTY720 dramatically inhibited neovascularization in tumor tissues. The half-life and AUClast are good for FTY720 in mice. FTY720 inhibited new vessel formation at cellular level. In addition, the microarray data showed the low-dose FTY720 (100 nM) induced the alteration of a few genes’ expression. Conclusion: FTY720 has potent antitumor activity both in vitro and in vivo, the mechanism of which is associated with proapoptotic, antiangiogenic and anti-inflammatory activities. The immune-suppressive activity of FTY720 may limit the antitumor activity in immune-competent individuals. This study provides a comprehensive understanding of the immune-suppressant FTY720 in antitumor. Note: This abstract was not presented at the conference. Citation Format: Zuoquan Xie, Xiaoxu Chen, Hui Xu, Jing Ai, Meiyu Geng. In vitro and In vivo antitumor activity of immune suppressant FTY720 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A83.

Purpose: Sphingosine-1-phosphate (S1P) is a bioactive lipid that promotes cell survival and proliferation, and modulation of lymphocyte egress. It plays important roles in the pathogenesis of inflammation and cancer. FTY720 is a “functional antagonist” of S1P by binding and induces the internalization and degradation of S1P receptors. FTY720 showed antitumor activity in several tumor models, but the mechanism has not been fully illustrated. In this study, we used microarray to explore the signaling pathways and genes regulated by FTY720, which may help to illustrate the mechanism of action. Experimental Procedures: We tested the inhibitory activity of FTY720 in various human cancer cell lines. Human colon cancer cells SW620, which are sensitive to FTY720, were selected and used for further study. SW620 was subcutaneously inoculated into Balb/c nude mice, then treated intraperitoneally with low dose (0.5 mg/kg/d) and high dose (5 mg/kg/d) of FTY720. Meanwhile, microarray analysis was used to explore the modulated genes by FTY720. Moreover, we tested the anti-neovascularization effect of FTY720 on endothelial cells (HMEC), which was shown to have this activity in vivo, and we also profiled the differentially expressed genes regulated by FTY720 on HMEC cells. Summary: FTY720 showed potent inhibitory activity on various cancer cell lines. The colon cancer cell SW620 was one of the most sensitive cells in response to FTY720 with an IC50 of 3.71 μM. FTY720 also inhibited new vessel formation at cellular level. In animal study, both low-dose and high-dose FTY720 dramatically inhibited the tumor growth in SW620 xenograft model. Since the serum concentration of FTY720 was much lower than its cellular cytotoxicity at low dose, we propose the antitumor activity may not only be due to a direct effect on tumor growth, but also on neovascularization and lowering of circulating lymphocytes. In transcriptomic study, we found cell cycle and DNA repair pathways were downregulated by FTY720 both in SW620 and HMEC cells. In HMEC cells, mTORC1, MYC, P53, FOXO1, and immune response pathways were downregulated while steroid metabolism and terpenoid backbone biosynthesis pathways were upregulated by FTY720. In SW620 cells, fatty acid metabolism, FOXO1, inflammatory and immune response pathways were downregulated while steroid metabolism and terpenoid backbone biosynthesis pathways were upregulated by FTY720. Thus, there are many overlaps of regulated pathways and genes by FTY720 between SW620 and HMEC cells. Interestingly, the angiogenesis and blood vessel morphogenesis were both downregulated by FTY772 in SW620 and HMEC cells. Conclusion: FTY720 has potent antitumor activity in vitro and in vivo. It showed pleiotropic effects on gene expression, especially downregulating those genes involved in cell cycle, angiogenesis, and inflammatory and immune response. This study displayed a landscape of differentially expressed genes modulated by FTY720 and provided a basis for further mechanistic study. Citation Format: Zuoquan Xie, Xiaoxu Chen, Qingzhong Zeng, Meiyu Geng. Transcriptomic landscape of the immune suppressant FTY720 in antitumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B61.

The interaction between tumor cells and their immunosuppressive microenvironment promotes tumor progression and drug resistance. Thus, simultaneously targeting tumor cells and stromal cells is expected to have synergistic antitumor effects. Herein, we present the preclinical antitumor investigation of 3D185, a novel dual inhibitor targeting FGFRs, which are oncogenic drivers, and CSF-1R, which is the major survival factor for protumor macrophages. The antitumor characteristics of 3D185 were assessed by a range of assays, including kinase profiling, cell viability, cell migration, immunoblotting, CD8+ T-cell suppression, and in vivo antitumor efficacy, followed by flow cytometric and immunohistochemical analyses of tumor-infiltrating immune cells and endothelial cells in nude mice and immune-competent mice. We found that 3D185 significantly inhibited the kinase activity of FGFR1/2/3 and CSF-1R, with equal potency and high selectivity over other kinases. 3D185 suppressed FGFR signaling and tumor cell growth in FGFR-driven models both in vitro and in vivo. In addition, 3D185 could inhibit the survival and M2-like polarization of protumor macrophages, reversing the immunosuppressive effect of macrophages on CD8+ T cells as well as CSF1-differentiated macrophage induced-FGFR3-aberrant cancer cell migration. Furthermore, 3D185 inhibited tumor growth via remodeling the tumor microenvironment in TAM-dominated tumor models. In summary, 3D185 is a dual inhibitor against FGFR1/2/3 and CSF-1R that exhibits potent antitumor activities. This compound is promising because it simultaneously targets tumor cells per se and the immunosuppressive tumor microenvironment to synergistically antagonize tumors. Our study provides a solid foundation for the investigation of 3D185 in cancer patients, particularly in patients with aberrant FGFR and abundant macrophages, who respond poorly to classic pan-FGFRi treatment. Now 3D185 is in phase I clinical trials. Citation Format: Yang Dai, Yichun Ji, Yanyan Shen, Yi Su, Bo Liu, Yueliang Wang, Deqiao Sun, Yuchen Jiang, Chuantao Zha, Zuoquan Xie, Jian Ding, Meiyu Geng, Jing Ai, Xia Peng, Pengcong Hou, Yi Chen. Preclinical evaluation of 3D185, a novel potent inhibitor of FGFR1/2/3 and CSF-1R, in FGFR-dependent and macrophage-dominant cancer models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A78.

Pyruvate dehydrogenase kinase PDK1 is a metabolic enzyme responsible for switching glucose metabolism from mitochondrial oxidation to aerobic glycolysis in cancer cells, a general hallmark of malignancy termed the Warburg effect. Herein we report the identification of JX06 as a selective covalent inhibitor of PDK1 in cells. JX06 forms a disulfide bond with the thiol group of a conserved cysteine residue (C240) based on recognition of a hydrophobic pocket adjacent to the ATP pocket of the PDK1 enzyme. Our investigations of JX06 mechanism suggested that covalent modification at C240 induced conformational changes at Arginine 286 through Van der Waals forces, thereby hindering access of ATP to its binding pocket and in turn impairing PDK1 enzymatic activity. Notably, cells with a higher dependency on glycolysis were more sensitive to PDK1 inhibition, reflecting a metabolic shift that promoted cellular oxidative stress and apoptosis. Our findings offer new mechanistic insights including how to therapeutically target PDK1 by covalently modifying the C240 residue. Cancer Res; 75(22); 4923–36. ©2015 AACR.

Triple-negative breast cancer (TNBC) is defined as a group of primary breast cancers lacking expression of estrogen, progesterone, and human epidermal growth factor receptor-2 (HER-2) receptors, characterized by higher relapse rate and lower survival compared with other subtypes. Due to lack of identified targets and molecular heterogeneity, conventional chemotherapy is the only available option for treatment of TNBC, but non-discordant positive therapeutic efficacy could not be achieved. Here, we demonstrated that these TNBC cells were sensitive to teriflunomide, which was a well-known immunomodulatory drug for treatment of relapsing multiple sclerosis (MS). Potent anti-cancer effects in TNBC in vitro, including proliferation inhibition, cell cycle delay, cell apoptosis, and suppression of cell motility and invasiveness, could be achieved with this agent. Of note, we showed that multiple signals involved in TNBC proliferation, survival, migratory, and invasive potential were under regulation by teriflunomide. Among them, we identified down-regulation of growth factor receptors to abolish growth maintenance, suppression of c-Myc, and cyclin D1 to contribute to its anti-proliferative effect, modulation of components of cell cycle to induce S-phase arrest, degradation of Bcl-xL, and up-regulation of BAX via activation of MAPK pathway to induce apoptosis, and inhibition of epithelial-mesenchymal transition (EMT) process, matrix metalloproteinase-9 (MMP9) expression, and inactivation of Src/FAK to reduce TNBC migration and invasion. The results identified teriflunomide may be of therapeutic benefit for the more aggressive and difficult-to-treat breast cancer subtype, indicating the use of teriflunomide for clinical trials for treatment of TNBC patients.

Background: Breast cancer is the most diagnosed female cancer and the second leading cause of cancer death in the United States. Triple negative breast cancer (TNBC), a special subtype, defined as breast cancer lacking estrogen, progesterone and HER-2 receptors, showed clinically aggressive features and was associated with poor prognosis. TNBC is resistant to endocrine or HER-2 targeted therapies, and only conventional chemotherapeutic regimens were accepted as the treatment guidelines. Therefore, searching for novel pharmaceutical agents for TNBC is urgent and a hot spot in present clinical research. Teriflunomide, an orally available immunomodulatory drug, approved for treatment of multiple sclerosis (MS) by FDA, has demonstrated the potential application in cancer therapy, such as chronic lymphocytic leukemia (CLL), prostate cancer and melanoma. Therefore, we assessed the therapeutic value of teriflunomide in TNBC cells. Methodology/Principal Findings: In this study, we showed that teriflunomide treatment resulted in a dose- and time-dependent inhibition of proliferation in three TNBC cell lines: MDA-MB-231, MDA-MB-468 and BT549. Meanwhile, the agent could also induce loss of clonogenic survival in dose-dependent fashion in TNBC cells. The analysis of cell cycle distribution by flow cytometry revealed that teriflunomide for 48 h entrapped TNBC cells in S-phase with concomitant reduction in both G1- and G2/M-phase. Furthermore, by Annexin-V/PI staining, we showed high doses of teriflunomide for 2 days led to significant necrosis and minor apoptosis in TNBC cells. Additionally, the effect of teriflunomide on TNBC cell migration and invasion was also tested using Boyden chamber assays. Short-term treatment of teriflunomide decreased the cell motility and invasiveness considerably in a concentration-dependent manner. When evaluated for underlying mechanisms, teriflunomide was found to modulate multiple cell signaling pathways in three TNBC cell lines. First, teriflunomide inhibited expression of proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Second, teriflumomide delayed cell cycle transition by up-regulating cyclin A, along with p27 down-regulation and unchanged cyclin B1. Third, teriflunomide regulated the cell survival proteins, such as up-regulation of BAX and down-regulation of Bcl-Xl, by activation of MAPK pathway. Fourth, teriflunomide suppressed the marker signals involved in epithelial–mesenchymal transition(EMT) and invasion and inhibited activation of FAK/Src complex. Fifth, teriflunomide down-regulated growth factor receptors involved in TNBC growth maintenance, such as EGFR, IGF1R and FGFR4. Conclusion/Significance: Teriflunomide, although an anti-inflammatory agent, is a potent inhibitor of TNBC cells through modulation of multiple signaling pathways and may be of therapeutic benefit for TNBC in clinical practice. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-21.

Objective: Growth factor receptor-binding protein 14, a new member of noncatalytic adaptor proteins family, has been shown to be upregulated in breast cancer. We investigated the prognostic value of growth factor receptor-binding protein 14 expression in breast cancer patients treated with neoadjuvant chemotherapy. Methods: Primary breast cancer specimens were taken from locally advanced breast cancer patients in a Phase II clinical trial of neoadjuvant chemotherapy and the expression pattern of growth factor receptor-binding protein 14 was determined by immunohistochemistry. Kaplan– Meier analysis and Cox regression model were used to assess disease-free and overall survival, according to the expression of growth factor receptor-binding protein 14 in tumor cells. Results: Our result showed that growth factor receptor-binding protein 14 was highly expressed in 23.1% of breast cancer sections, and high expression of growth factor receptor-binding protein 14 was significantly associated with better disease-free (P ¼ 0.016, hazard ratio 0.07, 95% confidence interval 0.06 –0 .08) and overall survival (P ¼ 0.004, hazard ratio 0.02, 95% confidence interval 0.02–0.03), compared with the low-expression group. Multivariate analysis indicated that high expression of growth factor receptor-binding protein 14 was an independent good prognostic factor for both disease-free (P ¼ 0.04, hazard ratio 0.37, 95% confidence interval 0.14 –0.98) and overall survival (P ¼ 0.03, hazard ratio 0.11, 95% confidence interval 0.10–0.82). Conclusions: High expression of growth factor receptor-binding protein 14 in breast cancer cells may help to identify low-risk patients for additional therapies after neoadjuvant chemotherapy.

Background: Clear cell renal cell carcinoma (ccRCC) is the most lethal form of kidney cancer. Small molecule VEGFR inhibitors are widely used but are not curative and various resistance mechanisms such as activation of the MET pathway have been described. Dual MET/VEGFR2 inhibitors have recently shown clinical benefit but limited preclinical data evaluates their effects in ccRCC. Methods: An interrogation of the Cancer Genome Atlas (TCGA) dataset was performed to evaluate oncogenic alterations in the MET/VEGFR2 pathway. We evaluated the in vitro effects of Cabozantinib, a dual MET/VEGFR2 inhibitor, using a panel of ccRCC cell lines. Drug effects of cell viability and proliferation, migration, cell scatter, anchorage independent growth, and downstream MET/VEGFR2 signaling pathways were assessed. Results: Twelve percent of TCGA cases had possible MET/HGF oncogenic alterations with co-occurrence noted (p

Oral squamous cell carcinoma (OSCC) comprises a subset of head and neck squamous cell carcinoma (HNSCC) with poor therapeutic outcomes and high glycolytic dependency. Neoadjuvant chemotherapy regimens of docetaxel, cisplatin and 5-fluorouracil (TPF) are currently accepted as standard regimens for HNSCC patients with a high risk of distant metastatic spread. However, the antitumor outcomes of TPF neoadjuvant chemotherapy in HNSCC remain controversial. This study investigated the role of lactate dehydrogenase B (LDHB), a key glycolytic enzyme catalyzing the inter-conversion between pyruvate and lactate, in determining chemotherapy response and prognosis in OSCC patients. We discovered that a high protein level of LDHB in OSCC patients was associated with a poor response to TPF regimen chemotherapy as well as poor overall survival and disease-free survival. Our in-depth study revealed that high LDHB expression conferred resistance to taxol but not 5-fluorouracil or cisplatin. LDHB deletion sensitized OSCC cell lines to taxol, whereas the introduction of LDHB decreased sensitivity to taxol treatment. Taxol induced a pronounced impact on LDHB-down-regulated OSCC cells in terms of apoptosis, G2/M phase cell cycle arrest and energy metabolism. In conclusion, our study highlighted the critical role of LDHB in OSCC and proposed that LDHB could be used as a biomarker for the stratification of patients for TPF neoadjuvant chemotherapy and the determination of prognosis in OSCC patients.

A771726, an orally available anti-inflammatory agent, has been approved for the treatment of multiple sclerosis by diminishing entire inflammatory responses through multiple signaling pathways. Recently, a few emerging studies have focused on the potential application of A771726 in cancer therapy, less on the treatment of breast cancer and particularly on overcoming drug resistance in breast cancer. We report here for the first time the cytotoxic activity and drug resistance reversal of A771726 in acquired tamoxifen-resistant breast cancer cell line MCF-7/LCC9. We discovered that A771726 treatment showed antiproliferative activities in MCF-7/LCC9 cells, which were even more sensitive to A771726 than their parental tamoxifen-sensitive cells (MCF‑7). A771726 also exerted pro-apoptotic activities and induced cell cycle arrest at the G1 phase. Notably, treatment of A771726 restored the sensitivity of MCF-7/LCC9 cells to tamoxifen. Western blot analysis revealed that A771726 decreased the phosphorylation level of Src, one key driver of tamoxifen resistance. Moreover, in order to comprehensively clarify the mechanisms of A771726 in anti-estrogen-resistant cells, we explored a genome-wide transcriptomic analysis, and showed that A771726 could modulate multiple signaling pathways (e.g. cell cycle, apoptosis, MAPK, metabolism and p53 signaling pathway) and cellular processes (e.g. signal transduction, transcription and cell cycle). Overall, our results indicate that A771726 alone and the combination of A771726 with anti-estrogens may be of therapeutic benefit for ER-positive and endocrine-resistant breast cancer.

Monocarboxylate transporter 4 (MCT4) is a cell membrane transporter of lactate. Recent studies have shown that MCT4 is over-expressed in various cancers; however, its role in cancer maintenance and aggressiveness has not been fully demonstrated. This study investigated the role of MCT4 in oral squamous cell carcinoma (OSCC), and found that it is highly expressed in OSCC patients by using immunohistochemistry. Moreover, this over-expression of MCT4 was closely associated with tumor size, TNM classification, lymphatic metastasis, distant metastasis and tumor recurrence, and also poor prognosis. To further study mechanisms of MCT4 in vitro, we used small-interfering RNA to silence its expression in OSCC cell lines. The results showed that knock-down of MCT4 decreased cell proliferation, migration, and invasion. The inhibition of proliferation was associated with down-regulation of p-AKT and p-ERK1/2, while decreased cell migration and invasion may be caused by down-regulation of integrin β4-SRC-FAK and MEK-ERK signaling. Together, these findings provide new insight into the critical role of MCT4 in cell proliferation and metastasis in OSCC.

Aberrant regulation of nuclear factor kappa B (NF-κB) transcription factor is involved in cancer development, progression and resistance to chemotherapy. JG3, a marine-derived oligomannurarate sulfate, was reported as a heparanase and NF-κB inhibitor to significantly block tumor growth and metastasis in various animal models. However, the detailed functional mechanism remains unclear. Here, we report that JG3 inhibits NF-κB activation by specifically antagonizing the doxorubicin-triggered Ataxia-telangiectasia-mutated kinase (ATM) and the sequential MEK/ERK/p90Rsk/IKK signaling pathway but does not interfere with TNF-α-mediated NF-κB activation. This selective inactivation of the specific NF-κB cascade is attributed to the binding capacity of JG3 for Mre11, a major sensor of DNA double-strand breaks (DSB). Based on this selective mechanism, JG3 showed synergistic effect with doxorubicin in a panel of tumor cells and did not affect immune system function as shown in the in vivo delayed-type hypersensitivity (DTH) and hemolysis assays. All these highlight the clinical potential of JG3 as a favorable sensitizer in cancer therapy. In addition, identification of Mre11 as a potential target in the development of NF-κB inhibitors provides a platform for the further development of effective anticancer agents.

Histone deacetylases (HDACs) play fundamental roles in the epigenetic regulation of gene expression and contribute to the growth, differentiation, and apoptosis of cancer cells. Although HDACs are recognized to be closely related to cancer development and altered expression of certain HDACs is observed in tumor samples, the arcane characters of HDACs in tumorigenesis have not been fully illustrated. Herein, we report that HDAC7 is a crucial player in cancer cell proliferation. Knockdown of HDAC7 resulted in significant G(1)/S arrest in different cancer cell lines. Subsequent investigations indicated that HDAC7 silencing blocked cell cycle progression through suppressing c-Myc expression and increasing p21 and p27 protein levels. The ectopic expression of c-Myc in turn antagonized the cell cycle arrest and repressed the elevation of p21 and p27 in HDAC7 silencing setting. Of note, HDAC7 deficiency was further identified to induce cellular senescence program, which was also reversed by c-Myc re-expression. Further chromatin immunoprecipitation assays indicated that HDAC7 directly binds with c-Myc gene and HDAC7 silencing decreased c-Myc mRNA level via reducing histone H3/H4 acetylation and repressing the association of RNA polymerase II (RNAP II) with c-Myc gene. Taken together, our findings highlight for the first time an unrecognized link between HDAC7 and c-Myc and offer a novel mechanistic insight into the contribution of HDAC7 to tumor progression.

509 Background: Sarcomatoid transformation in renal cell carcinoma (ccRCC) is one of the worst prognostic factors and outcome is extremely poor. We evaluate genetic alterations implicated in this process. Methods: Nephrectomy specimens from ccRCC with sarcomatoid transformation had DNA extracted from carcinoma, sarcomatoid, and normal kidney regions. Exome capture/Illumina sequencing was performed in 21 samples. Somatic mutation calling was accomplished by comparing sarcomatoid-normal and carcinoma-normal pairs. Results: Two tumors had evidence of hypermutation and a mutational signature consistent with mismatch repair deficiency. In the remaining tumors, 42.6% of somatic mutations were shared. Sarcomatoid regions had a greater mutation burden (p = 4.0x10-4). A low percentage (57.9%) of tumors had mutations in VHL. Mutations in ccRCC driver genes, including PBRM1, PTEN, SETD2, ARID1A, and BAP1, were common. All mutations in ARID1A and BAP1 were specific to sarcomatoid regions. A high percentage (31.5%) of TP53 mutations were observed, all specific to sarcomatoid regions and occurring with loss of heterozygosity. Lastly, mutations in genes not previously described in ccRCC were observed, most sarcomatoid-specific. These include genes implicated in cell adhesion, polarity, motility, and WNT signaling (FAT1, FAT2, FAT3, PTK7), retinoic acid-regulated cell differentiation (RQCD1, LRIF1), and ubiquitinated protein trafficking and cytokinesis (TSG101). Conclusions: Sarcomatoid transformation in ccRCC results from clonal divergence from a common somatic cell of origin. The sarcomatoid region has significantly greater mutational burden of known cancer driver genes. TP53 mutations occurred at a high frequency and were exclusive to the sarcomatoid region. Hypermutation is a unique characteristic observed in a subset of tumors. Additional cohorts and mechanistic studies are critical to elucidate the role of candidate driver alterations.

Background Selective estrogen receptor modulators, such as tamoxifen, play a pivotal role in the treatment of luminal-type breast cancer. However, in clinical applications, nearly half of breast cancer patients are insensitive to tamoxifen, a small number of whom have early recurrence or disease progression when receiving tamoxifen. The underlying mechanism of this resistance has not been determined. ZNF703 is a novel oncogene in the 15% of breast cancers that harbor 8p12 amplifications. Therefore, the goal of our study was to explore the role of ZNF703 in tamoxifen resistance. Methodology/Principal Findings We used immunohistochemistry techniques to examine ZNF703 expression in stage I-III primary breast cancer specimens and found a positive expression rate of 91.3%. All patients were divided into either high or low ZNF703 expression groups. We found that high ZNF703 expression mainly occurred in ER+ and PR+ breast cancers. Furthermore, 4-hydroxytamoxifen had different modes of action in breast cancer cell lines with high or low ZNF703 expression. ZNF703 overexpression in MCF-7 breast cancer cells activated the Akt/mTOR signaling pathway, downregulated ERα, and reduced the antitumor effect of tamoxifen. Low-dose tamoxifen did not suppress, but rather, stimulated the growth of cells overexpressing ZNF703. ZNF703 knockdown in MDA-MB-134 and HCC1500 luminal B-type breast cancer cell lines by siRNA significantly decreased survival rates when cells were treated with tamoxifen. Furthermore, targeting ZNF703 with a mTOR inhibitor increased the inhibitory effects of tamoxifen in ZNF703-overexpressing cells. Conclusion/Significance Our study suggests that ZNF703 expression levels may predict tamoxifen sensitivity. Tamoxifen should be administered with caution to those patients bearing tumors with ZNF703 overexpression. However, large clinical trials and prospective clinical studies are needed to verify these results.