guanosine-triphosphate and Carcinoma--Squamous-Cell

Cancer stem cells are thought to be responsible for rapid tumor growth, metastasis, and enhanced tumor survival following drug treatment. For this reason, there is a major emphasis on identifying proteins that can be targeted to kill cancer stem cells or control their growth, and transglutaminase type II (TGM2/TG2) is such a target in epidermal squamous cell carcinoma. TG2 was originally described as a transamidase in the extracellular matrix that crosslinks proteins by catalyzing ε-(γ-glutamyl)lysine bonds. However, subsequent studies have shown that TG2 is a GTP-binding protein that plays an important role in cell signaling and survival. In the present study, TG2 shows promise as a target for anticancer stem cell therapy in human squamous cell carcinoma. TG2 was determined to be highly elevated in epidermal cancer stem cells (ECS cells), and TG2 knockdown or suppression of TG2 function with inhibitors reduced ECS cell survival, spheroid formation, Matrigel invasion, and migration. The reduction in survival is associated with activation of apoptosis. Mechanistic studies, using TG2 mutants, revealed that the GTP-binding activity is required for maintenance of ECS cell growth and survival, and that the action of TG2 in ECS cells is not mediated by NF-κB signaling.. This study suggests that TG2 has an important role in maintaining cancer stem cell survival, invasive, and metastatic behavior and is an important therapeutic target to reduce survival of cancer stem cells in epidermal squamous cell carcinoma.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Enzyme Inhibitors; Epithelial Cells; Gene Knockdown Techniques; Genetic Vectors; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Neoplasm Invasiveness; Neoplastic Stem Cells; NF-kappa B; Protein Binding; Protein Glutamine gamma Glutamyltransferase 2; Signal Transduction; Transglutaminases

We have previously shown that E2F7 contributes to drug resistance in head and neck squamous cell carcinoma (HNSCC) cells. Considering that dysregulation of responses to chemotherapy-induced cytotoxicity is one of the major reasons for treatment failure in HNSCC, identifying the downstream effectors that regulate E2F7-dependent sensitivity to chemotherapeutic agents may have direct clinical impact. We used transcriptomic profiling to identify candidate pathways that contribute to E2F7-dependent resistance to doxorubicin. We then manipulated the expression of the candidate pathway using overexpression and knockdown in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and RacGAP1 in a custom tissue microarray (TMA) generated from HNSCC patient samples. Transcriptomic profiling identified RacGAP1 as a potential mediator of E2F7-dependent drug resistance. We validated E2F7-dependent upregulation of RacGAP1 in doxorubicin-insensitive SCC25 cells. Extending this, we found that selective upregulation of RacGAP1 induced doxorubicin resistance in previously sensitive KJDSV40. Similarly, stable knockdown of RacGAP1 in insensitive SCC25 cells induced sensitivity to doxorubicin in vitro and in vivo. RacGAP1 expression was validated in a TMA, and we showed that HNSCCs that overexpress RacGAP1 are associated with a poorer patient overall survival. Furthermore, E2F7-induced doxorubicin resistance was mediated via RacGAP1-dependent activation of AKT. Finally, we show that SCC cells deficient in RacGAP1 grow slower and are sensitized to the cytotoxic actions of doxorubicin in vivo. These findings identify RacGAP1 overexpression as a novel prognostic marker of survival and a potential target to sensitize SCC to doxorubicin.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cell Line, Tumor; Disease Models, Animal; Doxorubicin; Drug Resistance, Neoplasm; E2F7 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Mice; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; rac1 GTP-Binding Protein; rhoA GTP-Binding Protein

In this study we investigated the correlation between RhoC expression and cancer stem cells (CSCs) formation in head and neck squamous cell carcinoma (HNSCC). The inhibition of RhoC function was achieved using shRNA. The expression of stem cell surface markers, ALDH and CD44 were significantly low in two RhoC depleted HNSCC cell carcinoma cell lines. Furthermore, a striking reduction in tumorsphere formation was achieved in RhoC knockdown lines. The mRNA expression of RhoC in RhoC knockdown adherent and tumorspheres are dramatically down regulated as compared with the scrambled control. The mRNA expression of stem cell transcription factors; nanog, oct3/4 (Pouf1), and sox2 were significantly depleted in RhoC knockdown clones. Further, the phosphorylation of STAT3(ser727), and STAT3(tyr705) were significantly down regulated in RhoC knockdown clones. The overexpression of STAT3 in RhoC knockdown did not show any change in expression patterns of either-STAT3(tyr705) or stem cell transcription factors, signifying the role of RhoC in STAT3 activation and thus the expression of nanog, oct3/4 and sox2 in HNSCC. The expression of Inter leukin-6 (IL-6) in RhoC knockdown HNSCC cell lines was dramatically low as compared to the scrambled control. Further, we have shown a rescue in STAT3 phosphorylation by IL-6 stimulation in RhoC knockdown lines. This study is the first of its kind to establish the involvement of RhoC in STAT3 phosphorylation and hence in promoting the activation of core cancer stem cells (CSCs) transcription factors. These findings suggest that RhoC may be a novel target for HNSCC therapy.

Topics: Aldehyde Dehydrogenase 1 Family; Carcinoma, Squamous Cell; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Head and Neck Neoplasms; Humans; Hyaluronan Receptors; Interleukin-6; Isoenzymes; Neoplastic Stem Cells; Phosphorylation; Retinal Dehydrogenase; rho GTP-Binding Proteins; rhoC GTP-Binding Protein; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transcription Factors

Previously we showed that galanin, a neuropeptide, is secreted by human squamous cell carcinoma of the head and neck (SCCHN) in which it exhibits an autocrine mitogenic effect. We also showed that rap1, a ras-like signaling protein, is a critical mediator of SCCHN progression. Given the emerging importance of the galanin cascade in regulating proliferation and survival, we investigated the effect of GAL on SCCHN progression via induction of galanin receptor 2 (GALR2)-mediated rap1 activation. Studies were performed in multiple SCCHN cell lines by inducing endogenous GALR2, by stably overexpressing GALR2 and by downregulating endogenous GALR2 with siGALR2. Cell proliferation and survival, mediated by the ERK and AKT signaling cascades, respectively, were evaluated by functional and immunoblot analysis. The role of rap1 in GALR2-mediated proliferation and survival was evaluated by modulating expression. Finally, the effect of GALR2 on tumor growth was determined. GALR2 stimulated proliferation and survival via ERK and AKT activation, respectively. Knockdown or inactivation of rap1 inhibited GALR2-induced, AKT and ERK-mediated survival and proliferation. Overexpression of GALR2 promoted tumor growth in vivo. GALR2 promotes proliferation and survival in vitro, and promotes tumor growth in vivo, consistent with an oncogenic role for GALR2 in SCCHN.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; GTPase-Activating Proteins; Guanosine Triphosphate; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Receptor, Galanin, Type 2; RNA, Messenger; Transplantation, Heterologous; Tumor Burden

Grb2-associated binder 1 (GAB1) is a scaffold protein involved in numerous interactions that propagate signaling by growth factor and cytokine receptors. Here we explore in silico and validate in vivo the role of GAB1 in the control of mitogenic (Ras/MAPK) and survival (phosphatidylinositol 3-kinase (PI3K)/Akt) signaling stimulated by epidermal growth factor (EGF). We built a comprehensive mechanistic model that allows for reliable predictions of temporal patterns of cellular responses to EGF under diverse perturbations, including different EGF doses, GAB1 suppression, expression of mutant proteins, and pharmacological inhibitors. We show that the temporal dynamics of GAB1 tyrosine phosphorylation is significantly controlled by positive GAB1-PI3K feedback and negative MAPK-GAB1 feedback. Our experimental and computational results demonstrate that the essential function of GAB1 is to enhance PI3K/Akt activation and extend the duration of Ras/MAPK signaling. By amplifying positive interactions between survival and mitogenic pathways, GAB1 plays the critical role in cell proliferation and tumorigenesis.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; Feedback; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Biological; Transfection

Previous studies have demonstrated the presence in mouse epidermal tumors of a structurally and functionally altered ornithine decarboxylase (ODC). In this report, the enzymatic properties of ODC from normal human skin and squamous cell carcinomas are examined. Some tumors contained a more heat stable ODC than the enzyme found in normal skin. GTP stimulated enzyme activity in four of seven tumor extracts tested but had no effect on normal skin ODC. Kinetic analyses indicated that GTP either lowered the apparent Km of tumor ODC for L-ornithine, increased the Vmax, or had both effects, depending on the tumor examined. Gel filtration chromatography of crude tumor extracts indicated the existence of multiple molecular weight forms of ODC, some of which can be activated by GTP and some of which are unaffected by GTP. Some tumors contain both a GTP-activatable and -nonactivatable form of the enzyme. Immunolocalization studies demonstrated the presence within squamous cell carcinomas of cells with a constitutively high level of immunoreactive ODC, a situation never observed in normal skin tissue. These results suggest that some human squamous cell carcinomas contain a functionally altered ODC that may be aberrantly regulated.

Topics: Carcinoma, Squamous Cell; Enzyme Activation; Guanosine Triphosphate; Humans; Kinetics; Ornithine Decarboxylase; Skin; Skin Neoplasms

Biodistribution and tumor uptake studies were carried out with intravenously injected tracer doses of Gpp(NH)p labeled with 3H, 32P or 99mTc . Syrian golden hamsters with cheek pouch carcinomas, induced by repeated topical applications of DMBA, were used as a tumor model. The biodistributions of these three radionuclides were different, indicating significant molecular cleavage of this nucleotide analog. It was also apparent that this compound labeled with 99mTc may not be useful for tumor imaging due to low tumor-to-blood specific activity ratio. The cheek pouch carcinoma tumor model may be valuable for the evaluation of tumor localizing radiopharmaceuticals.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cricetinae; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Male; Mesocricetus; Mouth Neoplasms; Organotechnetium Compounds; Phosphorus Radioisotopes; Technetium; Tissue Distribution; Tritium