Squamous Cell Carcinoma Cells Research Articles

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.

The anti-tumour effect of induced pluripotent stem cells against submandibular gland carcinoma in rats is achieved via modulation of the apoptotic response and the expression of Sirt-1, TGF-β, and MALAT-1 in cancer cells.

The era of induced pluripotent stem cells (iPSCs) was used as novel biotechnology to replace embryonic stem cells bypassing the ethical concerns and problems of stem cell transplant rejection. The anti-tumour potential of iPSCs against many tumours including salivary cancer was proven in previous studies. The current study aimed to investigate the contribution of the Bax, Sirt-1, TGF-β, and MALAT genes and/or their protein expression to the pathogenesis of submandibular carcinogenesis before and after iPSCs treatment. Thirty Wistar albino rats were equally assigned into three groups: group I (control), group II (Squamous cell carcinoma (SCC)): submandibular glands were injected SCC cells, and group III (SCC/iPSCs): SCC rats were treated by 5 × 106 iPSCs. Submandibular gland sections were subjected to histological and immunohistochemical analyses to detect mucopolysaccharides, Bax, and TGF-β expression as well as PCR quantification for TGF-β, Sirt-1, and lncRNA MALAT-1 gene expressions. Western blotting was also used to detect Sirt-1 and TGF-β protein expressions. SCC group revealed infiltration by sheets of malignant squamous cells with or without keratin pearls and inflammatory cells, in addition to upregulation of TGF-β, Sirt-1, MALAT-1, and Bax, whereas SCC/iPSCs group showed an improved submandibular histoarchitecture with the maintenance of the secretory function. Bax and TGF-β immunoexpression were significantly reduced. The upregulated TGF-β, Sirt-1, and MALAT-1 genes were significantly decreased. iPSCs protected against the experimentally induced submandibular gland carcinoma that might be achieved via their regenerative potential and their regulatory modulation of Sirt-1, TGF-β, and MALAT-1 gene/protein expressions and of the apoptotic response in cancer cells.

Cancer-Associated Fibroblasts Facilitate Squamous Cell Carcinoma Lung Metastasis in Mice by Providing TGFβ-Mediated Cancer Stem Cell Niche.

Cancer-associated fibroblasts (CAFs) have been shown to enhance squamous cell carcinoma (SCC) growth, but it is unclear whether they promote SCC lung metastasis. We generated CAFs from K15.KrasG12D.Smad4–/– mouse SCCs. RNA expression analyses demonstrated that CAFs had enriched transforming growth factor-beta (TGFβ) signaling compared to normal tissue-associated fibroblasts (NAFs), therefore we assessed how TGFβ-enriched CAFs impact SCC metastasis. We co-injected SCC cells with CAFs to the skin, tail vein, or the lung to mimic sequential steps of lung metastasis. CAFs increased SCC volume only in lung co-transplantations, characterized with increased proliferation and angiogenesis and decreased apoptosis compared to NAF co-transplanted SCCs. These CAF effects were attenuated by a clinically relevant TGFβ receptor inhibitor, suggesting that CAFs facilitated TGFβ-dependent SCC cell seeding and survival in the lung. CAFs also increased tumor volume when co-transplanted to the lung with limiting numbers of SCC cancer stem cells (CSCs). In vitro, CSC sphere formation and invasion were increased either with co-cultured CAFs or with CAF conditioned media (which contains the highest TGFβ1 concentration) and these CAF effects were blocked by TGFβ inhibition. Further, TGFβ activation was higher in primary human oral SCCs with lung metastasis than SCCs without lung metastasis. Similarly, TGFβ activation was detected in the lungs of mice with micrometastasis. Our data suggest that TGFβ-enriched CAFs play a causal role in CSC seeding and expansion in the lung during SCC metastasis, providing a prognostic marker and therapeutic target for SCC lung metastasis.

Distinct immune microenvironment profiles of therapeutic responders emerge in combined TGF\u03b2/PD-L1 blockade-treated squamous cell carcinoma

Transforming growth factor beta (TGFβ) and programmed death-ligand 1 (PD-L1) are often overproduced in refractory squamous cell carcinoma (SCC). We examined spatial patterns of PD-L1+ cells in mouse and human SCCs and found that PD-L1 was primarily expressed on infiltrating leukocytes. Although combined TGFβ and PD-L1 blockade are undergoing cancer clinical trials, there are no predictive markers for therapeutic responders. To address this, we used both a small molecule TGFβ inhibitor in combination with anti-PD-L1 and a bifunctional fusion protein targeting both TGFβ and PD-L1 to treat mouse SCCs and found TGFβ inhibition enhanced PD-L1 blockade-induced tumor eradication in multiple tumor models. Furthermore, we identified distinct cell populations of responders and non-responders to bintrafusp alfa, with responders showing a shift toward a more immune-permissive microenvironment. The cellular and molecular signatures of responders versus non-responders to combined TGFβ and PD-L1 blockade provide important insights into future personalized immunotherapy in SCC.

Lung Cancer Screening Study in Macao Smoking Individuals

背景与目的中国澳门肺癌发病率逐年上升，吸烟人群是肺癌的高发人群，本研究旨在了解中国澳门长期吸烟人群的肺癌发病情况及胸部低剂量计算机断层扫描(low-dose computed tomography, LDCT)肺结节特点。方法通过澳门中华医学会会员私家医生推荐及宣传招募中国澳门无症状长期吸烟人士，行胸部LDCT检查，分析肺癌、肺部结节检出率及影像学特点。结果符合纳入条件者291例，检出肺癌10例，检出率3.44%(95%CI: 2.78%-4.01%)，其中，肺腺癌5例，鳞癌、小细胞肺癌各2例，腺鳞癌1例。早期肺癌4例，占40%。212例检出肺结节，肺结节总检出率72.9%(95%CI: 67.8%-78.0%); 疑似肺癌结节44例，检出率15.1%(95%CI: 11.0%-19.2%)。单发结节51例，无肺癌检出; 多发结节161例，检出肺癌9例，两组肺癌检出率无统计学差异(P > 0.05)。 < 6 mm实性结节与 < 5 mm非实性结节组168例，未检出肺癌; ≥6 mm实性结节与≥5 mm非实性结节组44例，检出肺癌9例，两组比较有统计学差异(P < 0.05)。结论长期吸烟人群中肺癌检出率高，类型以腺癌为主，肺部结节发生率高，当实性结节≥6 mm或非实性结节≥5 mm时，肺癌检出率增高。建议在符合高危因素的男性吸烟人群中推行胸部LDCT筛查肺癌，女性肺癌筛查，应重新界定高危因素。

Integrative Metabolomic and Lipidomic Profiling of Lung Squamous Cell Carcinoma for Characterization of Metabolites and Intact Lipid Species Related to the Metastatic Potential.

Simple SummaryNon-small cell lung cancer (NSCLC) is the most severe cancer showing a low 5-year survival rate of metastatic NSCLC, but there are few previous reports for prediction of metastatic potential and identification of therapeutic targets of lung squamous cell carcinoma (SQCC), a major type of NSCLC, with different metastatic potential based on metabolic and lipidomic profiling. We identified metabolites and intact lipid species relevant to lung SQCC metastatic potential, which could be applied to develop potential biomarkers and therapeutic targets.SQCC is a major type of NSCLC, which is a major cause of cancer-related deaths, and there were no reports regarding the prediction of metastatic potential of lung SQCC by metabolomic and lipidomic profiling. In this study, metabolomic and lipidomic profiling of lung SQCC were performed to predict its metastatic potential and to suggest potential therapeutic targets for the inhibition of lung SQCC metastasis. Human bronchial epithelial cells and four lung SQCC cell lines with different metastatic potentials were analyzed using gas chromatography–mass spectrometry and direct infusion-mass spectrometry. Based on the obtained metabolic and lipidomic profiles, we constructed models to predict the metastatic potential of lung SQCC; glycerol, putrescine, β-alanine, hypoxanthine, inosine, myo-inositol, phosphatidylinositol (PI) 18:1/18:1, and PI 18:1/20:4 were suggested as characteristic metabolites and intact lipid species associated with lung SQCC metastatic potential. In this study, we established predictive models for the metastatic potential of lung SQCC; furthermore, we identified metabolites and intact lipid species relevant to lung SQCC metastatic potential that may serve as potential therapeutic targets for the inhibition of lung SQCC metastasis.

Matrix Metalloproteinases (MMPs) in skin and oral pathology - Premalignant Diseases, Spinocellular Carcinoma and Basocellular Carcinoma (markers).

Matrix Metalloproteinases (MMPs) in skin and oral pathology - Premalignant Diseases, Spinocellular Carcinoma and Basocellular Carcinoma (markers).

MCL1 inhibition enhances the efficacy of docetaxel against airway-derived squamous cell carcinoma cells

MCL1 is an anti-apoptotic BCL2 family member that is often overexpressed in various malignant tumors. However, few reports have described the role of MCL1 in squamous cell carcinoma (SqCC) derived from airways including the lung. In this study, we examined whether MCL1 could be a novel druggable target for airway-derived SqCC, for which effective molecular targeted drugs are unavailable. We searched the Kaplan–Meier Plotter database and found that high MCL1 mRNA expression was significantly associated with shorter survival in patients with lower airway (lung) or upper airway (head and neck) derived SqCC. We also explored the Expression Atlas database and learned that authentic lung SqCC cell lines expressing both TP63 and KRT5 mRNA were extremely sparse among the publicly available “lung SqCC cell lines”, with an exception being HARA cells. HARA cells were highly dependent on MCL1 for survival, and MCL1-depleted cells were not able to grow, and even declined in number, upon docetaxel (DTX) exposure in vitro and in vivo. Similar in vitro experimental findings, including those in a 3D culture model, were also obtained using Detroit 562 pharyngeal SqCC cells. These findings suggested that combined treatment with MCL1 silencing plus DTX appears highly effective against airway-derived SqCC.

SLUG is upregulated and induces epithelial mesenchymal transition in canine oral squamous cell carcinoma.

SLUG, encoded by the Snai2 gene, is known to play a role in epithelial-mesenchymal transition (EMT), which contributes to cell invasion and metastasis in some types of human carcinomas. However, the mechanisms and roles of EMT in canine squamous cell carcinoma (SCC) have not yet been elucidated. We have previously established canine oral SCC cell lines, including tonsillar SCC, and in this study, we evaluated the effects of SLUG on the phenotypes regarding EMT of canine SCC cells. First, immunohistochemical analysis revealed that SLUG is upregulated in canine oral SCC tissues compared to that in non-tumoural oral mucosa. Furthermore, gain-of-function and loss-of-function of SLUG revealed that SLUG partly contributed to migration and invasion of cells, as well as the upregulation of EMT markers such as vimentin and SNAIL. Thus, the current study suggests that SLUG promotes cell migration and invasion through EMT induction in canine oral SCC, as well as human cancers.

HOPX Exhibits Oncogenic Activity during Squamous Skin Carcinogenesis

Cutaneous squamous cell carcinomas (SCCs) are frequent heterogeneous tumors arising from sun-exposed regions of the skin and characterized by complex pathogenesis. HOPX is a member of the homeodomain-containing superfamily of proteins holding an atypical homeodomain unable to bind to DNA. First discovered in the heart as a regulator of cardiac development, in the skin, HOPX modulates the terminal differentiation of keratinocytes. There is a particular interest in studying HOPX in squamous skin carcinogenesis because it has the atypical structure and the functional duality as an oncogene and a tumor suppressor gene, reported in different malignancies. In this study, we analyzed the effects of HOPX knockdown and overexpression on SCC tumorigenicity invitro and invivo. Our data show that HOPX knockdown in SCC cells inhibits their proliferative and invasive activity through the acceleration of apoptosis. We established that methylation of two alternative HOPX promoters leads to differential expression of HOPX transcripts in normal keratinocytes and SCC cells. Importantly, we report that HOPX acts as an oncogene in the pathogenesis of SCC probably through the activation of the second alternative promoter and the modulation of apoptosis.

Interplay and cooperation between SREBF1 and master transcription factors regulate lipid metabolism and tumor-promoting pathways in squamous cancer

Squamous cell carcinomas (SCCs) comprise one of the most common histologic types of human cancer. Transcriptional dysregulation of SCC cells is orchestrated by tumor protein p63 (TP63), a master transcription factor (TF) and a well-researched SCC-specific oncogene. In the present study, both Gene Set Enrichment Analysis (GSEA) of SCC patient samples and in vitro loss-of-function assays establish fatty-acid metabolism as a key pathway downstream of TP63. Further studies identify sterol regulatory element binding transcription factor 1 (SREBF1) as a central mediator linking TP63 with fatty-acid metabolism, which regulates the biosynthesis of fatty-acids, sphingolipids (SL), and glycerophospholipids (GPL), as revealed by liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics. Moreover, a feedback co-regulatory loop consisting of SREBF1/TP63/Kruppel like factor 5 (KLF5) is identified, which promotes overexpression of all three TFs in SCCs. Downstream of SREBF1, a non-canonical, SCC-specific function is elucidated: SREBF1 cooperates with TP63/KLF5 to regulate hundreds of cis-regulatory elements across the SCC epigenome, which converge on activating cancer-promoting pathways. Indeed, SREBF1 is essential for SCC viability and migration, and its overexpression is associated with poor survival in SCC patients. Taken together, these data shed light on mechanisms of transcriptional dysregulation in cancer, identify specific epigenetic regulators of lipid metabolism, and uncover SREBF1 as a potential therapeutic target and prognostic marker in SCC.

Abstract 3086: Decoding the intimate dialogues between stem cells and immune cell for cancer immunotherapy

Abstract Adult stem cells (SCs) reside in various body sites where they govern tissue homeostasis and orchestrate wound repair. As a group of long-lived and indispensable cells, they inevitably face the challenge of experiencing repeated bouts of inflammation initiated by tissue injury, or infection. Recently, a subset of TGF-β-reactive tumor-initiating cells were found at the tumor-stroma interface of in skin or head &amp; neck squamous cell carcinomas (SCC). These cells appear to be enriched with adult stem cells features. To prevent collateral damage from immune reactions in normal tissue or to drive oncogenic progression, it is essential for SCs or tumor initiating SCs (tSC) to develop unique capacity to adapt to inflammation. However, so far, it is still unclear how stem cells achieve immune adaptation. In order to explore this question, we designed a novel mouse model for spontaneous skin SCC that could be effectively challenged with adoptive T cell transfer (ACT)-based immunotherapy. Importantly, we genetically coupled the targeted antigen and HRASG12V, so that the tumors grown on these mice cannot easily silence the presentation of targeted antigen and upon ACT treatment, these tumors rapidly regress. Remarkably, singe cell RNA-seq followed by genetic lineage tracing revealed the ACT survivors were a group of TGFβ-responding tSCs. To determine the SC-specific programs that could be hijacked by cancer to drive immune evasion, we profiled these ACT-surviving tSCs, as well as normal hair follicle stem cells (HFSCs) that are isolated from highly inflammatory condition, such as a cutaneous wound. Surprisingly, in both normal SCs and tSCs, we observed activation of class II MHC and CD80, the immune modulatory molecules canonically found on antigen-presenting immune cells. To test the functional significance of these proteins for epithelial SCs, CD80 was ablated by designing an in vivo CRISPR gene editing strategy. Interestingly, when we profiled the immune composition of Cd80 cKO wound or tumor, we observed significant reduction in regulatory T cells (Treg), resulting drastic accumulation of neutrophils in the wounds of Cd80 cKO mice, or antigen-specific cytotoxic T cells in the skin SCC. Cancer and wounds share many characteristics, and cancer has been considered as “a wound that never heals”. Prior interpretations mainly pointed to the observations that similar molecular pathways drive stem cell proliferation, differentiation and stress responses in both wound and cancer. The intimate dialogue between stem cells and surrounding immune cells has long been overlooked. In this study, we have unveiled that the stem cell-immune cell crosstalk actually dictates the outcomes of successful tissue regeneration and this mechanism can be hijacked by cancer to survive from immunotherapy treatment. These discoveries will provide a new revenue to leverage and target these vital dialogues as novel cancer immunotherapy. Citation Format: Cynthia Truong, Weijie Guo, Alexandrer Rudensky, Elaine Fuchs, Yuxuan Phoenix Miao. Decoding the intimate dialogues between stem cells and immune cell for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3086.

Abstract LB213: ACTL6A (Actin-like Protein 6A) enhances the cancer phenotype

Abstract Epidermal squamous cell carcinoma (SCC) is an extremely common, invasive and metastatic cancer. ACTL6A, an important protein subunit of the SWI/SNF epigenetic regulatory complex, is frequently overexpressed in cancer cells. In the present study, we show that ACTL6A drives SCC cell proliferation, invasion, migration and tumor formation, and that this aggressive phenotype is markedly reduced in ACTL6A knockdown cells. Expression of ACTL6A is associated with reduced p21Cip1 level and treatment with ACTL6A-siRNA results in a marked increase in p21Cip1 and attenuation of the cancer phenotype. Molecular studies reveal that ACTL6A suppresses p21Cip1 promoter activity to reduce mRNA and p21Cip1 protein and that loss of p21Cip1 expression appears to be biologically meaningful, as p21Cip1 knockdown enhances the aggressive phenotype. ACTL6A suppression of p21Cip1 expression requires the p53 and Sp1 transcription response elements in the p21Cip1 gene promoter. In addition, ACTL6A knockdown results in increased p53 tumor suppressor expression. These studies show that ACTL6A facilitates an aggressive cancer phenotype by reducing p53 and p21Cip1 mRNA and protein and suggests that ACTL6A contributes to an aggressive cancer phenotype in epidermal squamous cell carcinoma. Citation Format: Suruchi Shrestha, Gautam Adhikary, Wen Xu, Sivaveera Kandaysamy, Richard L. Eckert. ACTL6A (Actin-like Protein 6A) enhances the cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB213.

Abstract 269: Harnessing the mechanical power of nanomachines to treat cancer: Light-activated molecular nanomachines kill melanoma and oral cancer cells

Abstract Introduction: Molecular nanomachines (MNMs) have been studied for their anti-tumor efficacy in a number of solid malignancies. They consist of small (1 nm) organic molecule-based motors that change conformation upon light activation and mechanically drill holes in the cell membrane resulting in cellular necrosis. That opens a novel way of molecular mechanical therapy. Recently, we generated a blue light spectrum (395-405 nm)-activated MNMs which showed promising results in pancreatic cancer cell lines. Herein, we evaluate the in vitro efficacy in various oral and skin cancers including squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma, a disease characterized by fast progression and therapeutic resistance. Methods: MNMs activated by the blue light spectrum were synthesized. In vitro efficacy of the activated MNMs (8 µM in 0.1% DMSO) was investigated in skin cancer cell lines. Cells were incubated with MNMs for 30 minutes, then treatment groups were subjected to 150 mW/cm2 blue light for 4 minutes or 300 mW/cm2 for 5 min. Control groups included MNMs without light, 0.1% DMSO (required for solubility of the MNMs) with light, and 0.1% DMSO without light. Cell viability was quantitatively evaluated utilizing propidium iodide (PI) and flow cytometry assay with each datapoint representing triplicates. Additionally, cells were grown and tested by clonogenic assay 1d and 9d following light and MNMs treatment. Results: B16-F10 (murine melanoma cancer cell line) treated with light-activated MNMs exhibited almost 100% loss in viability when treated with MNMs and light. Solution only (0.1% DMSO), unactivated MNMs and DMSO solution combined with light exhibited less than 5% change in cell viability suggesting that all the controls are non-cytotoxic evaluated by PI-flow cytometry at 3 h after treatment. Clonogenic assay 9 day post treatment confirmed 100% cell death for the MNMs + light treated cells, while showing nearly 80-90% survival of cells in the light only treatment control. Other treated cell lines include SCC cell lines (ROC 1 and ROC 3) showing 100% loss in cell viability when treated with MNMs and light. Conclusions: Light-activated MNM exhibit profound in vitro efficacy in multiple skin cancer cells lines. MNMs are a promising novel therapeutic modality for the treatment of oral and skin cancers, and there are no known resistance mechanisms for nanomechanical therapy. They are now being tested on murine tumor models of B16-F10 in C57BL/6 mice and further preclinical characterization of antitumor efficacy and tolerability are necessary in these studies. Citation Format: Ciceron Ayala-Orozco, Alexis van Venrooy, Dongdong Liu, Yewen Shi, Jacob Beckham, David Izhaky, James M. Tour, Jeffrey N. Myers, Roberto Rangel. Harnessing the mechanical power of nanomachines to treat cancer: Light-activated molecular nanomachines kill melanoma and oral cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 269.

Abstract LB212: The VGLL4 tumor suppressor inhibits transglutaminase-2 dependent YAP1-TEAD signaling to attenuate the cancer phenotype

Abstract Epidermal squamous cell carcinoma (SCC) is among the most common and aggressive forms of cancer. We previously showed that the pro-cancer transcription factor, YAP1, drives an aggressive SCC cancer phenotype by binding to nuclear TEAD transcription factors to activate pro-cancer gene expression. Vestigial-like protein 4 (VGLL4) is a tumor suppressor that is downregulated in epithelial cancers. It binds to TEAD factors to displace YAP1 binding to reduce YAP1-dependent transcription and target gene expression. The present study shows that VGLL4 overexpression reduces SCC cell TEAD-dependent transcription to reduce expression of mRNA and protein encoding YAP1-target genes (CTGF, CYR61, CCND1), and pro-cancer collagen genes (COL1A2, COL3A1) leading to reduced cell proliferation, spheroid formation, invasion and migration. Identical results were observed in response to transient and stable VGLL4 overexpression. Similarly, VGLL4 expression reduces YAP1 activity in tumors leading to reduced YAP1 target and pro-cancer collagen gene expression and reduced and EMT marker proteins levels which is associated with reduced tumor formation. Loss of YAP1 responsive gene expression appears to be essential for VGLL4 attenuation of the aggressive phenotype, as forced overexpression of CCND1 or COL1A2 antagonizes VGLL4 action. These findings suggest VGLL4 functions as a tumor suppressor in SCC by repressing YAP1/TEAD signaling and consequently attenuating the SCC phenotype. Citation Format: McKayla B. Mickle, Gautam Adhikary, Wen Xu, Suruchi Shrestha, Richard L. Eckert. The VGLL4 tumor suppressor inhibits transglutaminase-2 dependent YAP1-TEAD signaling to attenuate the cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB212.

HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

Human populations of Black African ancestry have a relatively high risk of aggressive cancer types, including keratinocyte‐derived squamous cell carcinomas (SCCs). We show that primary keratinocytes (HKCs) from Black African (Black) versus White Caucasian (White) individuals have on average higher oncogenic and self‐renewal potential, which are inversely related to mitochondrial electron transfer chain activity and ATP and ROS production. HSD17B7 is the top‐ranked differentially expressed gene in HKCs and Head/Neck SCCs from individuals of Black African versus Caucasian ancestries, with several ancestry‐specific eQTLs linked to its expression. Mirroring the differences between Black and White HKCs, modulation of the gene, coding for an enzyme involved in sex steroid and cholesterol biosynthesis, determines HKC and SCC cell proliferation and oncogenicity as well as mitochondrial OXPHOS activity. Overall, the findings point to a targetable determinant of cancer susceptibility among different human populations, amenable to prevention and management of the disease.

Imaging of Oral SCC Cells by Raman Micro-Spectroscopy Technique.

We used Raman micro-spectroscopy technique to analyze the molecular changes associated with oral squamous cell carcinoma (SCC) cells in the form of frozen tissue. Previously, Raman micro-spectroscopy technique on human tissue was mainly based on spectral analysis, but we worked on imaging of molecular structure. In this study, we evaluated the distribution of four components at the cell level (about 10 μm) to describe the changes in protein and molecular structures of protein belonging to malignant tissue. We analyzed ten oral SCC samples of five patients without special pretreatments of the use of formaldehyde. We obtained cell level images of the oral SCC cells at various components (peak at 935 cm−1: proline and valine, 1004 cm−1: phenylalanine, 1223 cm−1: nucleic acids, and 1650 cm−1: amide I). These mapping images of SCC cells showed the distribution of nucleic acids in the nuclear areas; meanwhile, proline and valine, phenylalanine, and amide I were detected in the cytoplasm areas of the SCC cells. Furthermore, the peak of amide I in the cancer area shifts to the higher wavenumber side, which indicates the α-helix component may decrease in its relative amounts of protein in the β-sheet or random coil conformation. Imaging of SCC cells with Raman micro-spectroscopy technique indicated that such a new observation of cancer cells is useful for analyzing the detailed distribution of various molecular conformation within SCC cells.

Expression of Neurokinin B Receptor in the Gingival Squamous Cell Carcinoma Bone Microenvironment.

Gingival squamous cell carcinoma (SCC) frequently invades the maxillary or mandibular bone, and bone destruction is known as a key prognostic factor in gingival SCCs. Recently, Neurokinin 3 receptor (NK-3R), the receptor ligand for NK-3, which is a member of the tachykinin family expressed in the central nervous system, was identified through pathway analysis as a molecule expressed in osteoclasts induced by the hedgehog signal. Although the expression of NK-3R has been detected in osteoclast and SCC cells at the bone invasion front, the relationship between NK-3R expression and the prognosis of gingival SCC patients remains unclear. In the present study, we retrospectively reviewed 27 patients with gingival SCC who had undergone surgery with curative intent. Significantly higher NK-3R expression in tumor cells was found in a case of jawbone invasion than in a case of exophytic poor jawbone invasion. On the other hand, no significant association was observed between NK-3R tumor-positive cases and tumor size, TNM stage, or tumor differentiation. The survival rate tended to be lower in NK-3R tumor-positive cases, but not significantly. However, the disease-specific survival rate was significantly lower in patients with a large number of NK-3R-positive osteoclasts than in those with a small number of them at the tumor bone invasion front. Our results suggest that NK-3R signaling in the gingival SCC bone microenvironment plays an important role in tumor bone destruction and should be considered a potential therapeutic target in advanced gingival SCC with bone destruction.

RSPO1-mutated fibroblasts from non-tumoural areas of palmoplantar keratoderma display a cancer-associated phenotype.

R-spondin (RSPO)1 is a fibroblast-secreted protein that belongs to the R-spondin protein family which is essential for reproductive organ development, epithelial stem cell renewal and cancer induction or suppression. RSPO1 gene mutations cause palmoplantar hyperkeratosis with squamous cell carcinoma (SCC) of the skin, 46XX sex reversal and true hermaphroditism. To characterize RSPO1-deficient skin fibroblasts derived from two patients with mutations in RSPO1, with palmoplantar hyperkeratosis, recurrent SCC and 46XX sex reversal, to provide further insight into disease-related skin tumourigenesis. Fibroblast cultures from non-tumoural palmoplantar skin biopsies were established to evaluate features and properties that may be altered at cancer onset, i.e. proliferation, extracellular matrix contraction and invasion, as well as TGF-β and matrix metalloproteinase (MMP) secretion. Fibroblasts demonstrated increased proliferative potential in vitro, a high level of collagen contraction and invasion by SCC cells, release of high levels of pro-inflammatory and pro-fibrotic TGF-β, and increased expression of MMP1 and MMP3. Analysis of the expression of selected proteins associated with RSPO1-activated pathways confirmed sustained activation of the TGF-β signalling pathway and indicated a loss of TGF-β inhibitory feedback. Also, treatment of fibroblasts with a recombinant RSPO1 protein aggravated this pro-inflammatory phenotype, suggesting caution in designing therapeutic strategies based on restoration of protein function. Our findings indicate that fibroblasts from RSPO1-mutated patients behave similarly to cancer-associated fibroblasts. Chronic inflammation and fibrotic changes in palmoplantar skin may play a role in SCC development and recurrence, possibly by irreversibly activating the tumourigenic phenotype of fibroblasts.

Heparin-Binding Protein 17/Fibroblast Growth Factor-Binding Protein-1 Knockout Inhibits Proliferation and Induces Differentiation of Squamous Cell Carcinoma Cells.

Simple SummaryFibroblast growth factor (FGF) plays an important role in tumor growth by inducing angiogenesis in addition to promoting the proliferation of squamous cell carcinoma (SCC) and oral squamous cell carcinoma (OSCC) cells. Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) purified from A431 cell-conditioned media based on its capacity to bind to FGF-1 and FGF-2 is recognized as a pro-angiogenic molecule as a consequence of its interaction with FGF-2. In this study, we have examined the functional role of HBp17/FGFBP-1 in A431 and HO-1-N-1 cells using the CRISPR/Cas9 technology. Our results showed that HBp17/FGFBP-1 knockout inhibited cell proliferation, colony formation, and cell motility compared to control. The amount of FGF-2 was decreased in culture medium conditioned by HBp17/FGFBP-1 knockout cells compared to control. We performed cDNA/protein expression analysis followed by Gene Ontology and protein–protein interaction analysis. The results demonstrate that both gene and protein expression related to epidermal development, cornification, and keratinization were upregulated in HBp17/FGFBP-1-knockout A431 and HO-1-N-1 cells.Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) has been observed to induce the tumorigenic potential of epithelial cells and is highly expressed in oral cancer cell lines and tissues. It is also recognized as a pro-angiogenic molecule because of its interaction with fibroblast growth factor (FGF)-2. In this study, we examined the functional role of HBp17/FGFBP-1 in A431 and HO-1-N-1 cells. Originally, HBp17/FGFBP-1 was purified from A431 cell-conditioned media based on its capacity to bind to FGF-1 and FGF-2. We isolated and established HBp17/FGFBP-1-knockout (KO)-A431 and KO-HO-1-N-1 cell lines using the clusters of regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing technology. The amount of FGF-2 secreted into conditioned medium decreased for A431-HBp17-KO and HO-1-N-1-HBp17-KO cells compared to their WT counterparts. Functional assessment showed that HBp17/FGFBP-1 KO inhibited cell proliferation, colony formation, and cell motility in vitro. It also inhibited tumor growth in vivo compared to controls, which confirmed the significant difference in growth in vitro between HBp17-KO cells and wild-type (WT) cells, indicating that HBp17/FGFBP-1 is a potent therapeutic target in squamous cell carcinomas (SCC) and oral squamous cell carcinomas (OSCC). In addition, complementary DNA/protein expression analysis followed by Gene Ontology and protein–protein interaction (PPI) analysis using the Database for Visualization and Integrated Discovery and Search Tool for the Retrieval of Interacting Genes/Proteins showed that both gene and protein expression related to epidermal development, cornification, and keratinization were upregulated in A431-HBp17-KO and HO-1-N-1-KO cells. This is the first discovery of a novel role of HBp17/FGFBP-1 that regulates SCC and OSCC cell differentiation.