Human Squamous Cell Carcinoma Cell Lines Research Articles

Anti-cancer effects of green synthesized gold nanoparticles using leaf extract of Annona muricata. L against squamous cell carcinoma cell line 15 through apoptotic pathway

Background: Oral cancer remains one of the most dreadful diseases in developing nations. Currently, there has been a rise in the prevalence of tongue squamous cell carcinoma (SCC), with a poor prognosis. The use of standard treatment approaches against oral cancer patients brings about several side effects. In recent years, nanomedicine has provided a versatile platform for developing new targeted therapeutic modalities. However, safety remains a concern in the synthesis of nanoparticles (NPs). Therefore, the present study aims to synthesize safer phytoconstituent-mediated gold NPs (AuNPs) utilizing leaf extracts of Annona muricata, where the biochemical components of the plant leaf act as the reducing and capping agents in the synthesis of NPs, and to evaluate its anti-cancer activity against SCC. Materials and Methods: In this in vitro experimental study, AuNPs were synthesized through an effective, simple, and ecologically sound green synthesis method. After characterization of these synthesized AuNPs, in vitro assays such as 3-(4, 5-dimethylthiazole2-yl)-2, 5-biphenyl tetrazolium bromide, wound healing, and clonogenic assays were carried out to investigate the anti-cancer potential of green synthesized AuNPs in the human tongue SCC cell line (SCC-15), and the possible mechanism of action was evaluated through gene and protein expression analysis of Bax, Bcl-2, and p53 genes. The results were expressed as mean ± standard deviation using Statistical Package for Social Sciences (SPSS) 20.0 software and Student's t-test was performed for experimental data. P ≤0.05 were considered statistically significant. Results: The in vitro assays demonstrated that the synthesized AuNPs are exhibiting anti-cancer activity by apoptosis of SCC-15 cells in a dose-dependent manner. Further, it also revealed a highly significant decrease in anti-apoptotic Bcl-2 gene expression, whereas pro-apoptotic genes p53 and Bax revealed a highly significant increase, which is statistically significant compared to the control (P < 0.05). Conclusion: Our findings demonstrated that the AuNPs synthesized from A. muricata leaf extract could act as a novel anticancer agent, particularly against SCC, after further scrutiny.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 transfection of guide RNA targeting on MMP9 as anti-cancer therapy in human cutaneous squamous cell carcinoma cell line A431.

Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin malignancy, representing around 20% of all skin cancers. It is the main cause of death due to non-melanoma skin cancer every year. Metastatic cutaneous SCC is associated with poor prognosis in patients and warrants a more effective and specific approach such as disruption of genes associated with cancer metastasis. Matrix metalloproteinases (MMPs) are enzymes involved in cancer progression and are regarded as major oncotargets. Among others, MMP9 plays critical roles in tumour progression, angiogenesis, and invasion of cutaneous SCC. We aimed to determine whether the MMP9 gene is a suitable gene target for anti-cancer therapy for cutaneous SCC. We performed clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 transfection of guide RNA (gRNA) targeting the MMP9 gene into human cutaneous SCC cell line A431. Following CRISPR transfection treatment, the viability (p < 0.01) and migratory activities (p < 0.0001) of in vitro cutaneous SCC cells were found to be reduced significantly. The use of quantitative polymerase chain reaction (qPCR) also revealed downregulation of the mRNA expression levels of cancer-promoting genes TGF-β, FGF, PI3K, VEGF-A, and vimentin. Direct inhibition of the MMP9 gene was shown to decrease survivability and metastasis of cutaneous SCC cell line A431. Our findings provided direct evidence that MMP9 is important in the viability, proliferation, and metastasis of cutaneous SCC cells. It serves as a positive foundation for future CRISPR-based targeted anti-cancer therapies in treating skin cancer and other forms of malignancies that involve MMPs as the key determinants.

Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models.

Simple SummaryOral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2)-dependent mechanism. We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain.Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.

Abstract 501: Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis

Abstract Introduction: Heavy alcohol (EtOH) drinking and tobacco smoking increase the risk for upper digestive (e.g. oral and esophageal) squamous cell carcinomas (SCC) in individuals with polymorphic mutations in aldehyde dehydrogenase 2 (Aldh2). Mutant Aldh2E487K protein delays mitochondrial clearance of acetaldehyde, the chief metabolite of EtOH and constituents of tobacco smoke, aka a major human carcinogen. The role of Aldh2 in tumor biology remains elusive. Methods: Single cell-derived three-dimensional (3D) organoids were generated from murine oral and esophageal preneoplastic and SCC lesions carrying wild-type Aldh2WT or mutant Aldh2E487K as well as human SCC cell lines with Aldh2E487K, the latter subjected to xenograft transplantation in immunodeficient mice. 3D organoids and xenograft tumors were analyzed by morphology and flow cytometry to determine CD44, autophagy (cyto-ID), proliferation (EdU), and reactive oxygen species (ROS) following EtOH exposure ex vivo (0.1-2% EtOH) or in vivo (10% EtOH in drinking water) along with or without inhibitors of alcohol metabolism (4-methylpyrazole; 4MP, siRNA against Adh1B and Cyp2E1), and autophagy flux (chloroquine; CQ or siRNA against Atg7). Flow cytometry was performed to determine cancer stem-like cells with high CD44 expression (CD44H cells). Results: EtOH stimulated xenograft tumor growth and increased highly proliferative intratumoral CD44H cells with increased autophagy. EtOH-stimulated tumor growth and induction of CD44H cells were sharply suppressed by concurrent 4MP treatment, indicating that EtOH is directly metabolized in SCC cells to promote tumor growth via CD44H cells. These EtOH-induced effects were recapitulated in 3D organoids where Aldh2E487K augmented EtOH-mediated induction of CD44H cells and organoid formation with increased cellular atypia. EtOH induced mitochondrial superoxide and ROS-dependent autophagy activation in CD44H cells. Inhibition of autophagy prevented EtOH from enriching CD44H cells in 3D organoids, suggesting that autophagy may limit alcohol-induced oxidative stress as a cytoprotective mechanism. Conclusions: These studies provide not only mechanistic insights into the role of Aldh2 mutation in oral and esophageal SCC pathogenesis but also establish the utility of the 3D organoid system to test the functional interplay between genetic and environmental factors which may subsequently be targeted for pharmacological intervention in the setting of personalized medicine. Citation Format: Masataka Shimonosono, Takashi Kijima, Hisatsugu Maekawa, Satoshi Takada, Koji Tanaka, Varun Sahu, Manti Guha, Andres J. Klein-Szanto, J. Alan Diehl, Adam J. Bass, Anil K. Rustgi, Elizabeth Philipone, Angela J. Yoon, Hiroshi Nakagawa. Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 501.

155 A biomarker function of HMGA2 in cutaneous squamous cell carcinoma development

The high mobility group AT-hook 2 (HMGA2) gene encodes a transcription factor that is expressed during embryonic development but down-regulated in adult tissues. Re-expression of HMGA2 in adult tissues is often associated with both benign and malignant tumor formation. HMGA2 has been identified as a biomarker of melanoma progression and prognosis, but its role in non-melanoma skin cancer development remains controversial. In this study, we measured HMGA2 expression in normal human keratinocytes and found a significant decrease in HMGA2 mRNA expression in adult keratinocytes compared to neonatal keratinocytes. HMGA2 is highly expressed in human cutaneous squamous cell carcinoma (SCC) cell lines and primary human SCC tumors, but not detected in adjacent normal skin. In mouse skin, Hmga2 has been found to be translocated from the cell membrane into the nucleus during DMBA/TPA-induced skin tumorigenesis. While Hmga2 expression is absent in the hair follicle in non-UV-irradiated mouse, UV irradiation increased Hmga2 expression in both the epidermis and hair follicles. In agreement, we found enhanced Hmga2 expression in UV-induced mouse skin SCCs and also Hmga2 reactivation in the dermis from UV-irradiated mice. Furthermore, oncogene depletion such as FOXM1 and TRIP13 in human SCC cell lines using CRISPR/Cas9 decreased the expression of HMGA2 coupled with decreased cell proliferation and survival. Taken together, these results demonstrate that the upregulation of HMGA2 is an important biomarker of skin SCC tumorigenesis. Moreover, understanding of the mechanism for HMGA2 overexpression in skin hair follicle cells can develop the new drug for skin cancer treatment.

TGF-β1 induces N-cadherin expression by upregulating Sox9 expression and promoting its nuclear translocation in human oral squamous cell carcinoma cells.

Squamous cell carcinoma (SCC) is the most frequent cancer that develops in the oral cavity. Epithelial-mesenchymal transition (EMT) is known to play an important role in the process of metastasis of SCC cells. In our previous study, we demonstrated that TGF-β1 induced EMT in the human oral SCC (hOSCC) cell line HSC-4. We also found that Slug plays an important role in suppressing E-cadherin expression and promotion of the migratory activity of HSC-4 cells. However, we also demonstrated that Slug does not participate in upregulation of N-cadherin expression, suggesting that EMT-related transcription factors other than Slug also play an important role in the process. In the present study, we aimed to elucidate how the transcription factor Sox9 affects the TGF-β1-induced upregulation of N-cadherin expression in HSC-4 cells. We found that TGF-β1 upregulated Sox9 expression in HSC-4 cells. In addition, Sox9 siRNA significantly abrogated the TGF-β1-induced upregulation of N-cadherin expression and inhibited the TGF-β1-promoted migratory activity in HSC-4 cells. We also demonstrated that TGF-β1 upregulated the phosphorylation status of Sox9 and then promoted nuclear translocation of Sox9 from the cytoplasm, possibly resulting in an increase in N-cadherin expression. The cyclic AMP-dependent protein kinase A inhibitor H-89, which is known to suppress phosphorylation of Sox9, significantly abrogated the TGF-β1-induced upregulation of N-cadherin expression. These results suggested that TGF-β1 induced N-cadherin expression by upregulating Sox9 expression and promoting its nuclear translocation, which results in EMT progression in hOSCC cells.

Troglitazone, a Selective Ligand for PPARγ, Induces Cell-cycle Arrest in Human Oral SCC Cells.

We attempted to clarify the role of Peroxisome proliferator-activated receptor γ (PPARγ) and its ligand, troglitazone (TRO) on oral squamous cell carcinoma (SCC). The expression of PPARγ gene was examined in 47 human oral SCC tissues and two human oral SCC cell lines, CA9-22 and HSC-4. The effects of TRO on the growth and cell-cycle progression of human oral SCC cells were examined. PPARγ mRNA was detected in 20 of 47 oral SCC tissues and two human oral SCC cells. TRO significantly suppressed the growth of the cells, but did not induce apoptosis. CA9-22 cells treated with TRO showed an increased fraction in the G1 phase and decreased fractions in the S and G2-M phases. TRO did not induce apoptosis in oral SCC cells, but did inhibit the growth of the cells by arresting the cell cycle at G1 phase.

Molecular Profiling of Keratinocyte Skin Tumors Links Staphylococcus aureus Overabundance and Increased Human β-Defensin-2 Expression to Growth Promotion of Squamous Cell Carcinoma

The skin microbiota plays a prominent role in health and disease; however, its contribution to skin tumorigenesis is not well understood. We comparatively assessed the microbial community compositions from excision specimens of the main human non-melanoma skin cancers, actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Keratinocyte skin tumors are characterized by significantly different microbial community compositions, wherein AK and SCC are more similar to each other than to BCC. Notably, in SCC, which represents the advanced tumor entity and frequently develops from AK, overabundance of Staphylococcus aureus, a known skin pathogen, was noted. Moreover, S. aureus overabundance was significantly associated with increased human β-defensin-2 (hBD-2) expression in SCC. By challenging human SCC cell lines with S. aureus, a specific induction of hBD-2 expression and increased tumor cell growth was seen. Increased proliferation was also induced by directly challenging SCC cells with hBD-2. Together, our data indicate that a changed microbial community composition in SCC, specified by S. aureus overabundance, might promote tumor cell growth via modulation of hBD-2 expression.

Sunlight, Vitamin D, and Xeroderma Pigmentosum.

Sunlight, in particular UV-B radiation, is an important factor for endogenous vitamin D production as 80-90% of the required vitamin D needs to be photosynthesized in the skin. The active form of vitamin D, vitamin D3 or calcitriol, binds to the ligand-activated transcription factor vitamin D receptor (VDR) for genomic and non-genomic effects. Recently, calcitriol and analogs have been shown to have antiproliferative effects in mouse and human BCC and SCC cell lines in vitro. As UV radiation plays a critical role in the photosynthesis of vitamin D, stringent sun protection, as recommended for xeroderma pigmentosum (XP) patients, may impact their vitamin D levels.XP is a rare autosomal recessive disorder with a worldwide prevalence of 1 in 1,000,000. XP can be divided into seven different complementation groups: XP-A to XP-G. The complementation groups correspond with the underlying gene defect. Defects in these genes lead to a defective nucleotide excision repair (NER), which is necessary to remove UV-induced DNA damage such as the UV photoproducts cyclobutane pyrimidine dimers (CPD) and 6-4 pyrimidine-pyrimidone (6-4 PP) dimer. Additionally, a variant form with a mutation in the translational polymerase η gene (PolH), also called XP variant (XPV), exists. Patients with XPV show a defect in translesion synthesis. Due to their inability to repair UV-induced lesions, XP patients exhibit an increased risk for UV-induced nonmelanoma skin cancer (NMSC) such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) as well as melanoma. Although no curative therapy for XP exists today, numerous options for the treatment and prophylaxis of skin cancer have become available.

436 Macrophages promote growth of squamous cancer independent of T cells

We assessed the role of macrophages in squamous cell carcinoma (SCC) behavior. We used mouse SCC cells derived from tumors harboring a Kras12D activation mutation and Smad4 deletion in keratin 15 (K15)-positive stem cells, and a human SCC cell line, FaDu. SCC cells were transplanted into immune-compromised or -competent (syngeneic) recipients. After tumors were established, we profiled tumor infiltrating leukocytes using CyTOF. CD45+ leukocytes comprised 10-25% of the total tumor cells, and tumor-associated macrophages (TAMs) were 5-10% of the CD45+ cells. There were typically 2-3 fold more M2 TAMs than M1 TAMs. We used clodronate liposomes to deplete TAMs. We found that the number of TAMs was not affected by the presence of T cells but differed among tumors derived from different SCC lines. Clodronate significantly reduced TAMs and macrophages in the spleen, resulting in reduced SCC volumes. Tumors with clodronate treatment did not show decreased proliferation, but exhibited increased apoptosis and reduced vascular density. FLIP (Fas-associated via death domain-like interleukin-1β converting enzyme inhibitory protein), an apoptosis inhibitor abundantly produced in tumor cells and TAMs, was reduced in tumor cells of clodronate treated mice. Reduced FLIP levels correlated with reductions in phosphorylated nuclear NFkB p65 and NFkB inhibitor attenuated FLIP protein levels in SCC cells. Further, TGFb1 serum levels and pSmad3 were reduced in clodronate-treated mice, but their reductions were insufficient to reverse epithelial-mesenchymal transition (EMT) or TGFb-mediated angiogenesis in endothelial cells. Consequently, metastasis was not significantly reduced by macrophage reduction. However, reduced pSmad3 correlated with reduction of its transcriptional target, vascular endothelial growth factor A (VEGFA), in clodronate-treated tumor cells. Taken together, our study revealed that macrophages contribute to SCC expansion through interactions with tumor cells but are dispensable for SCC metastasis. Our study provides novel insights into understanding the contributions and limitations of TAMs in SCC progression.

NRG1 is a critical regulator of differentiation in TP63-driven squamous cell carcinoma.

Squamous cell carcinomas (SCCs) account for the majority of cancer mortalities. Although TP63 is an established lineage-survival oncogene in SCCs, therapeutic strategies have not been developed to target TP63 or it's downstream effectors. In this study we demonstrate that TP63 directly regulates NRG1 expression in human SCC cell lines and that NRG1 is a critical component of the TP63 transcriptional program. Notably, we show that squamous tumors are dependent NRG1 signaling in vivo, in both genetically engineered mouse models and human xenograft models, and demonstrate that inhibition of NRG1 induces keratinization and terminal squamous differentiation of tumor cells, blocking proliferation and inhibiting tumor growth. Together, our findings identify a lineage-specific function of NRG1 in SCCs of diverse anatomic origin.

Liposomal Avicequinone-B formulations: Aqueous solubility, physicochemical properties and apoptotic effects on cutaneous squamous cell carcinoma cells

BackgroundAvicequinone-B (Naphtho[2,3-b]furan-4,9-dione) is a furanonaphthoquinone derivative. It is a hydrophobic compound with poor aqueous solubility, which may restrict its potential pharmaceutical and biomedical applications. PurposeWe synthesized different liposomal formulations of Avicequinone-B, and measured their particle size, aqueous solubility, and physicochemical properties. In addition, we investigated the anticancer activity of liposomal Avicequinone-B in human cutaneous squamous cell carcinoma (SCC) cells. MethodsLiposomal Avicequinone-B formulations were synthesized using the thin-film hydration method. Drug yield, encapsulation efficiency and aqueous solubility were determined by high performance liquid chromatography. Particle size and polydispersity index were measured by submicron particle size analyzer, and ultrastructural morphology was visualized by transmission electron microscopy. Thermal transitions were determined by differential scanning calorimetry. Anti-skin cancer activity was determined in HSC-1 cells (human cutaneous SCC cell line) using the MTS cytotoxicity assay, apoptosis was assessed by caspase-3/7 activity assay, mitochondrial membrane potential was determined by JC-10 assay, and signal transduction pathways were evaluated by Western blot analysis. ResultsLiposomal Avicequinone-B formulations showed adequate yield and high encapsulation efficiency. These liposomal formulations produced small, uniformly sized nanoparticles, and greatly increased the aqueous solubility of Avicequinone-B. Differential scanning calorimetry showed loss of thermal phase transitions. In addition, liposomal Avicequinone-B showed significant cytotoxic effect on HSC-1 cells, through reduction of mitochondrial membrane potential, increased cytosolic cytochrome-c level, increased cleaved caspase 8 level, and induction of apoptosis. This was mediated through activation of ERK, p38 and JNK signaling pathways. ConclusionLiposomal Avicequinone-B demonstrated improved aqueous solubility and physicochemical characteristics, and induced apoptosis in cutaneous SCC cells. Therefore, liposomal Avicequinone-B may have potential uses as a topical anti-skin cancer drug formulation in the future.

TWEAK/Fn14 Interaction Confers Aggressive Properties to Cutaneous Squamous Cell Carcinoma

Recent studies showed that TWEAK/Fn14 signaling participates in the progression of internal malignancies. However, its role in the biological properties of cutaneous squamous cell carcinoma (SCC) remains unclear. This study was designed to explore the effect of TWEAK/Fn14 activation on cutaneous SCC as well as the relevant mechanism. The expression of TWEAK and Fn14 was determined in tissue samples of patients with cutaneous SCC. Human primary keratinocytes and SCC cell lines were cultured invitro, receiving stimulation of TWEAK. The xenografts of SCC were generated subcutaneously in BALB/c nude mice. The results showed that both TWEAK and Fn14 were highly expressed in human cutaneous SCC. Moreover, TWEAK/Fn14 activation promoted the proliferation, migration, and invasion of cultured SCC cells. Interestingly, TNFR2 was upregulated in cultured SCC cells, and the transfection of TNFR2 small interfering RNA abrogated the effect of TWEAK on these cells. Finally, the favorable effect of TWEAK/Fn14 signals was confirmed in BALB/c nude mice with SCC xenografts. In conclusion, TWEAK/Fn14 signals contribute to the progression of cutaneous SCC, possibly involving the TNF-α-independent TNFR2 signal transduction.

PUB045 Anti-Tumor Effect of Honokiol in Lung Squamous Cell Carcinoma

Patients with lung squamous cell carcinoma (SCC) still benefit few from new therapies of lung cancer, and seeking for potential effective therapies is imperative. Honokiol (HNK) is a natural compound isolated from the magnolia plant with numerous pharmacological activities and exerts anticancer activity. Here, we demonstrated that the effect of HNK in lung SCC cells in vitro and related signaling mechanisms. Cell viability of human lung SCC cell lines NCI-H520 and SK-MES-1 exposed to HNK were measured by CCK-8. Flow cytometry was applied to identify the cellular apoptosis with HNK exposure of SCC cells compared to lung adenocarcinoma cells. Fibroblast growth factor(FGF)-2 expression of both SCC cells with HNK exposure were explored by quantitative real-time PCR. Furthermore, the effect of HNK on FGFR1 and signal pathways related to proliferation, apoptosis and cell cycle were detected by western blot. In two subpopulations of human lung SCC cells, NCI-H520 and SK-MES-1, HNK exposure remarkably inhibited the cell proliferation in both time-and dose-dependent manners. Compared to A549 cells, a more significant cell apoptosis was observed in NCI-H520 cells after HNK incubation. Next, real-time PCR analysis indicated that HNK down-regulated the expression of FGF2 in both NCI-H520 cells and SK-MES-1 cells in dose dependence. Meanwhile, HNK also obviously inhibited FGFR1 in both cell lines. Moreover, HNK exposure activated PARP and caused caspase-3 cleavage, suppressed cyclineD1 and ERK expression. These results broaden our understanding of the mechanisms on HNK effects in lung SCC, and reinforce the possibility of its potential anticancer benefit on SCC patients.

GLI1 Blockade Potentiates the Antitumor Activity of PI3K Antagonists in Lung Squamous Cell Carcinoma.

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K-mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448-59. ©2017 AACR.

AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma.

Aquaporin 3 (AQP3) and phospholipase D2 (PLD2) are abnormally expressed and/or localized in squamous cell carcinoma (SCC). AQP3 transports glycerol to PLD2 for the synthesis of lipid second messenger, which can mediate the effect of the AQP3/PLD2 signaling module in the regulation of keratinocyte proliferation and differentiation. However, the role of the AQP3/PLD2 signaling module in the pathogenesis of SCC remains to be fully elucidated. In the present study, the expression levels of AQP3 and PLD2 in tissue samples were examined using immunohistochemistry, it was found that the expression levels of AQP3 and PLD2 in tissue samples of actinic keratosis (AK), Bowen's disease (BD) and SCC were significantly increased. AQP3 small interfering RNA (siRNA) and PLD2 siRNA were constructed and used for transfection into the human A431 SCC cell line, and their anticancer effect on SCC was examined. The mRNA expression and protein expression levels of AQP3 and PLD2 were significantly downregulated following siRNA transfection. AQP3 siRNA and PLD2 siRNA inhibited the proliferation and promoted the apoptosis of A431 cells. Taken together, the findings of the present study suggested that increased levels of AQP3 and PLD2 were correlated with tumor progression and development in SCC. AQP3 siRNA and PLD2 siRNA significantly downregulated the mRNA and protein levels of AQP3 and PLD2 in the A431 cells; inhibiting proliferation and promoting apoptosis in vitro. The concomitant effects of AQP3/PLD2 signaling by inhibiting the expression of siRNA may be important for the treatment of SCC in the future.

Abstract 3017: Non-classical activation of GLI1 as a therapeutic target for squamous cell lung cancer

Abstract The Hedgehog (Hh) signaling pathway is critical for embryonic developmental processes and its deregulation is implicated in a wide variety of tumor types. However, the role of the Hh signaling pathway in the initiation and growth of non-small cell lung cancer is largely unknown. The purpose of this study is to investigate the role of GLI1, a major Hh pathway transcription factor, in lung squamous cell carcinoma (SCC) and to test the therapeutic potential of targeting GLI1. GLI1 expression in human SCC cell lines was evaluated by quantitative PCR and Western Blot. siRNA and shRNA of GLI1 in these cell lines were utilized in vitro and in vivo to test the requirement of GLI1 in tumor growth. Small molecule modulators of GLI1 were tested for their therapeutic potential. We have demonstrated that GLI1 has a critical role in SCC progression. GLI1 expression was significantly elevated in lung SCC compared to normal lung and lung adenocarcinoma patient specimens in several human genomic databases. Importantly, overexpression of GLI1 was correlated with poor overall survival in lung cancer patients. siRNA-mediated knock down of GLI1 in SCC cell lines decreased the expression of GLI1 target genes and caused a significant reduction in colony formation. Stable knock down of GLI1 in SCC cell lines caused a significant reduction in growth of xenograft tumors indicating the critical role of GLI1 in lung SCC progression. Inhibition or activation of SMO, an upstream component of Hh pathway, did not change GLI1 expression level in SCC cell lines. However, inhibition of PI3K/mTOR and MAPK signaling pathways down regulated GLI1 expression. These results suggested that GLI1 expression is dependent on PI3K/mTOR and MAPK pathway activity rather than Hh ligand. PI3K/mTOR inhibitor, or arsenic trioxide (ATO), a direct inhibitor of GLI proteins, significantly reduced GLI1 expression, proliferation, and clonogenicity in SCC cell lines. We have also observed a significant growth reduction in SCC xenografted tumors treated with combination of PI3K inhibitor and ATO. Our findings suggest that GLI1 is essential for lung SCC tumor progression. Furthermore, GLI1 expression in SCC is independent of Hh pathway ligand action and dependent on MAPK and PI3K pathway activity. Direct inhibition of GLI1 by repurposing ATO in combination with a PI3K inhibitor may represent a novel therapeutic strategy for lung SCC. Citation Format: Sahba Kasiri, Chunli Shao, Baozhi Chen, Alexandra Wilson, Paul Yenerall, Brenda Timmons, patrick dospoy, Suzie Hight, Luc Girard, Hui Tian, Carmen Behrens, Ignacio Wistuba, Adi Gazdar, James Kim. Non-classical activation of GLI1 as a therapeutic target for squamous cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3017.

104 Melanoma associated antigen A3 (MAGE-A3) influences proliferation of metastatic squamous cell carcinoma (SCC) in vitro

104 Melanoma associated antigen A3 (MAGE-A3) influences proliferation of metastatic squamous cell carcinoma (SCC) in vitro A Santana, N Roudiani, J Therrien, JH Lee, D Felsen and J Carucci 1 Dept. of Derm., NYU Langone Med. Ctr., New York, NY, 2 Stiefel Laboratories, Research Triangle Park, NC and 3 Inst. for Ped. Urol., Weill Cornell Medicine, New York, NY In order to identify novel targets for cancer immune therapy, our lab has profiled the expression of cancer testis antigens (CTA) in primary human SCC. CTAs are immunogenic and may be suitable targets for immunotherapy, or as biomarkers for aggressive biological behavior. We recently showed that MAGE-A3 is highly expressed in aggressive SCC with poor prognosis. We hypothesized that MAGE-A3 may be a novel target for immunotherapy and/or a biomarker for metastatic SCCs. In this study we determined MAGE-A3 expression in and the effect of antibody treatment on growth of human (A431) and murine (Pam 212 and LY2, BALB/c) SCC cell lines. Pam 212 cells develop locally invasive SCC in syngeneic hosts and LY2 were isolated from a rare Pam 212 metastasis (Yuspa et al., JID, 1983; Dong et al., J Cell Biochem 1997). A431, Pam 212, and LY2 cell lines express MAGE-A3 at the transcript and protein level as measured by qRT-PCR and immunoblot, respectively. Moreover, A431 SCC growth and proliferation was significantly inhibited after MAGE-A3 antibody treatment measured by MTT (P 0.001) and crystal violet (P 0.001). We found that cell growth and proliferation of locally invasive Pam 212 cells was unaffected by anti-MAGE-A3 treatment. However, metastatic tumor derived LY2 cell growth and proliferation was significantly inhibited after MAGE-A3 antibody treatment measured by MTT (P 0.01) and crystal violet (P 0.01). MAGE-A3 expression could potentially serve both as a cancer biomarker and vaccine candidate due to its expression by SCC cells in vitro. Interestingly, MAGE-A3 antibody treatment inhibited the growth of A431 and LY2, SCCs that metastasize in vivo. These results suggest that metastatic SCC may rely on signaling events downstream of MAGE-A3 to support proliferation. Ongoing studies focus on elucidation of pathways involved in MAGE driven SCC proliferation in vivo using immune competent BALB/c mice. 105 Keratinocyte p38a loss results in increased tumor initiation, decreased malignant progression, and altered tumor type specification during two-stage chemical carcinogenesis in murine skin A Kiss, AC Koppel, C Cataisson, J Anders, S Yuspa, P Bible, M Kellett, MI Morasso and T Efimova 1 Anatomy and Regenerative Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, 2 Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD and 3 Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD The p38a signaling pathway not only regulates the inflammation and stress responses, but also controls, in a context-specific manner, the homeostatic balance of cell proliferation, differentiation and survival, deregulation of which is known to contribute to tumor development. Studies in mouse models revealed both tumor-suppressing and tumor-promoting roles of p38a, depending on tissue and tumor type, and tumor stage. However, the in vivo functional contributions of p38a to the skin carcinogenesis remain incompletely understood. We explored the in vivo role of p38a in two-stage chemical skin carcinogenesis using mice with conditional epidermal keratinocyte-specific genetic ablation of p38a (p38a-cKO). Mutant mice exhibited a significantly increased tumor multiplicity, and, in addition to characteristic benign and malignant squamous tumors, developed benign well-differentiated sebaceous adenomas containing a signature activating Hras mutation. Only 4% of tumors in p38a-cKO mice underwent malignant conversion to squamous cell carcinomas (SCCs), compared with 38% of tumors in wild-type (WT) mice (p<0.0001). p38a deficiency resulted in a significantly reduced size and a more differentiated morphology of p38a-cKO SCCs, relative to WT SCCs. RNA sequencing revealed p38a-dependent transcriptional changes in genes involved in cell differentiation, development, proliferation, cell death, and inflammation, both in control and in v-ras-transformed keratinocytes. Altogether, our data reveal novel roles for p38a signaling in control of tumor initiation, malignant progression, and tumor type specification.

Functional significance of transgelin-2 in uterine cervical squamous cell carcinoma.

Transgelin-2 (TAGLN2) has previously been found to be highly expressed in uterine cervical squamous cell carcinoma (SCC) tissues by proteomic analyses. The present study investigated the role of TAGLN2 in the malignant behaviors of cervical SCC cells in vitro and in vivo, and the clinical significance of TAGLN2 using immunohistochemistry for human cervical SCC tissues. Antisense (AS) constructs of TAGLN2 cDNA (AS clones) and the empty vector (control clone) were transfected into a human uterine SCC cell line (SKG IIIa), and malignant behaviors were analyzed in vitro. In an in vivo experiment, 10(7) cells of the AS and control clones were subcutaneously inoculated into female BALB/c nude mice. In immunohistochemistry with anti-TAGLN2 antibodies for human cervical SCC, FIGO stage IA and IB (n = 75), the expression patterns of TAGLN2 were divided into two groups: weak and strong. The relation between expression pattern and prognosis was analyzed. Suppression of TAGLN2 inhibited cancer cell migration and secretion of matrix metalloproteinases. Tumors in the control clone group continued to grow, whereas those in the AS clone group clearly stopped growing. Six weeks after injection, the tumor size was significantly smaller in the AS clone group than in the control clone group. Immunohistochemistry revealed that the strong pattern was associated with poor overall survival compared with the weak pattern by the Kaplan-Meier method. TAGLN2 plays functional roles in the progression of cervical SCC. Suppression of TAGLN2 may be a new strategy for the treatment of cervical SCC.

Nonclassical activation of gli1 as a therapeutic target for squamous cell lung cancer

Background: The Hedgehog (Hh) signaling pathway is critical for embryonic development and its deregulation is implicated in a number of tumor types. The role of the Hh signaling pathway, however, in the initiation and growth of non-small cell lung cancer is largely unknown. Here, we investigate the role of the Hh pathway transcription factor, GLI1, in lung squamous cell carcinoma (SCC) and as a potential therapeutic target for treatment of lung SCC. Methods: GLI1 expression in human SCC cell lines was evaluated by quantitative PCR and Western Blot. siRNA and shRNA of GLI1 in these cell lines were utilized in vitro and in vivo to test the requirement of GLI1 in tumor growth. Small molecule modulators of GLI1 were tested for their therapeutic potential. Results: GLI1 mRNA expression was significantly elevated in lung SCC compared to normal lung and lung adenocarcinoma patient specimens in several human genomic databases. Importantly, overexpression of GLI1 was correlated with poor overall survival in lung cancer patients. siRNA-mediated knock down of GLI1 in SCC cell lines decreased the expression of GLI1 target genes and caused a significant reduction in colony formation. Stable knock down of GLI1 in SCC cell lines caused a significant reduction in growth of xenograft tumors indicating the critical role of GLI1 in lung SCC progression. Inhibition or activation of SMO, an upstream component of Hh pathway, did not alter GLI1 expression level in lung SCC cell lines. However, inhibition of PI3K/AKT and MAPK signaling pathways down-regulated GLI1 expression, suggesting that GLI1 expression is dependent on PI3K/AKT and MAPK pathway activity rather than Hh ligand. Small molecule inhibition of PI3K/ mTOR pathway or GLI1 significantly reduced GLI1 expression, proliferation, and clonogenicity in SCC cell lines. Combinatorial inhibition of PI3K and GLI1 by BKM120 and arsenic trioxide (ATO), respectively, significantly abrogated the in vivo growth SCC tumors in mice and correlated with decreased tumor GLI1 expression. Conclusion: Our findings demonstrate that GLI1 is essential for lung SCC tumor progression. Furthermore, GLI1 expression in SCC is independent of Hh pathway ligand action and dependent on MAPK and PI3K pathway activity. Direct inhibition of GLI1 by repurposing ATO in combination with a PI3K inhibitor may represent a novel therapeutic strategy for lung SCC.