Model Of Squamous Cell Carcinoma Research Articles

Cutaneous squamous cell carcinoma characterized by MALDI mass spectrometry imaging in combination with machine learning

Cutaneous squamous cell carcinoma (SCC) is an increasingly prevalent global health concern. Current diagnostic and surgical methods are reliable, but they require considerable resources and do not provide metabolomic insight. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables detailed, spatially resolved metabolomic analysis of tissue samples. Integrated with machine learning, MALDI-MSI could yield detailed information pertaining to the metabolic alterations characteristic for SCC. These insights have the potential to enhance SCC diagnosis and therapy, improving patient outcomes while tackling the growing disease burden. This study employs MALDI-MSI data, labelled according to histology, to train a supervised machine learning model (logistic regression) for the recognition and delineation of SCC. The model, based on data acquired from discrete tumor sections (n = 25) from a mouse model of SCC, achieved a predictive accuracy of 92.3% during cross-validation on the labelled data. A pathologist unacquainted with the dataset and tasked with evaluating the predictive power of the model in the unlabelled regions, agreed with the model prediction for over 99% of the tissue areas. These findings highlight the potential value of integrating MALDI-MSI with machine learning to characterize and delineate SCC, suggesting a promising direction for the advancement of mass spectrometry techniques in the clinical diagnosis of SCC and related keratinocyte carcinomas.

Interleukin 33 supports squamous cell carcinoma growth via a dual effect on tumour proliferation, migration and invasion, and T cell activation

Interleukin (IL)-33 is an important cytokine in the tumour microenvironment; it is known to promote the growth and metastasis of solid cancers, such as gastric, colorectal, ovarian and breast cancer. Our group demonstrated that the IL-33/ST2 pathway enhances the development of squamous cell carcinoma (SCC). Conversely, other researchers have reported that IL-33 inhibits tumour progression. In addition, the crosstalk between IL-33, cancer cells and immune cells in SCC remains unknown. The aim of this study was to investigate the effect of IL-33 on the biology of head and neck SCC lines and to evaluate the impact of IL-33 neutralisation on the T cell response in a preclinical model of SCC. First, we identified epithelial and peritumoural cells as a major local source of IL-33 in human SCC samples. Next, in vitro experiments demonstrated that the addition of IL-33 significantly increased the proliferative index, motility and invasiveness of SCC-25 cells, and downregulated MYC gene expression in SCC cell lines. Finally, IL-33 blockade significantly delayed SCC growth and led to a marked decrease in the severity of skin lesions. Importantly, anti-IL-33 monoclonal antibody therapy increase the percentage of CD4+IFNγ+ T cells and decreased CD4+ and CD8+ T cells secreting IL-4 in tumour-draining lymph nodes. Together, these data suggest that the IL-33/ST2 pathway may be involved in the crosstalk between the tumour and immune cells by modulating the phenotype of head and neck SCC and T cell activity. IL-33 neutralisation may offer a novel therapeutic strategy for SCC.

Line-field confocal optical coherence tomography for in vivo visualization of morphological characteristics of squamous cell carcinoma in a murine model.

Non-invasive imaging with line-field confocal optical coherence tomography (LC-OCT) can support the diagnosis of squamous cell carcinoma (SCC) through visualization of morphological characteristics specific to skin cancer. We aimed to visualize prominent morphological characteristics of SCC using LC-OCT in a well-established murine SCC model. Nine hairless mice were exposed to ultraviolet radiation three times weekly for 9 months to induce SCC development. Visible SCC tumors (n = 9) were imaged with LC-OCT and the presence of 10 well-described morphological characteristics of SCC were evaluated in the scans by two physicians with adjudication by a third. Overall, murine morphological characteristics resembled corresponding features previously reported in human SCCs. Interrupted dermal-epidermal junction occurred in 100% of tumors. In epidermis, the most frequently observed characteristics were severe epidermal dysplasia (100%) and tumor budding (89%). Common dermal characteristics included broad strands (100%) and collagen alterations (78%). LC-OCT imaging can be used to non-invasively visualize morphological characteristics specific to SCC in an in vivo preclinical model.

Inducible nitric oxide synthase (iNOS)-activated Cxcr2 signaling in myeloid cells promotes TGFβ-dependent squamous cell carcinoma lung metastasis

Transforming growth factor beta (TGFβ) activity is linked to metastasis in many cancer types, but whether TGFβ activity is necessary for squamous cell carcinoma (SCC) lung metastasis has not been studied. Here we used a lung metastatic SCC model derived from keratin 15 (K15). KrasG12D.Smad4−/− SCC and human SCC specimens to identify metastasis drivers and test therapeutic interventions. We demonstrated that a TGFβ receptor (TGFβR) inhibitor reduced lung metastasis in mouse SCC correlating with reduced CD11b+/Ly6G+ myeloid cells positive for inducible nitric oxide synthase (iNOS). Further, TGFβ activity and iNOS were higher in primary human oral SCCs with metastasis than SCCs without metastasis. Consistently, either depleting myeloid cells with anti-Gr1 antibody or inhibiting iNOS with L-N6-(1-iminoethyl)-l-lysine (L-NIL) reduced SCC lung metastasis. L-NIL treated tumor-bearing mice exhibited reductions in tumor-infiltrating myeloid cells and in plasma Cxcl5 levels, and attenuated primary tumor growth with increased apoptosis and decreased proliferation. Blocking Cxcl5 with an antagonist of its receptor Cxcr2, SB225002, also reduced SCC lung metastasis.

Updating the biologically based dose-response model for the nasal carcinogenicity of inhaled formaldehyde in the F344 rat.

Chronic inhalation of formaldehyde by F344 rats causes nasal squamous cell carcinoma (SCC). This outcome is well-characterized: including dose-response and time course data for SCC, mechanistic endpoints, and nasal dosimetry. Conolly et al. (Toxicol. Sci. 75, 432-447, 2003) used these resources to develop a biologically based dose-response (BBDR) model for SCC in F344 rats. This model, scaled up to humans, has informed dose-response conclusions reached by several international regulatory agencies. However, USEPA concluded that uncertainties precluded its use for cancer risk assessment. Here, we describe an updated BBDR model that addresses uncertainties through refined dosimetry modeling, revised analysis of labeling index data, and an extended dataset where both inhaled (exogenous) and endogenous formaldehyde (exogF, endoF) form DNA adducts. Further, since Conolly et al. (ibid) was published, it has become clear that, when controls from all F344 inhalation bioassays are considered, accounting for over 4000 rats, at most one nasal SCC occurred. This low spontaneous incidence constrains possible contribution of endoF to the formation of nasal SCC via DNA reactivity. Further, since both exogF and endoF form DNA adducts, this constraint also applies to exogF. The revised BBDR model therefore drives SCC formation through the cytotoxicity of high concentration exogF. An option for direct mutagenicity associated with DNA adducts is retained to allow estimation of an upper bound on adduct mutagenicity consistent with the lack of a spontaneous SCC incidence. These updates represent an iterative refinement of the 2003 model, incorporating new data and insights to reduce identified model uncertainties.

PAK4 kinase as a potential therapeutic target of the transcription factor p63-meditated squamous cell carcinomas of the pancreas.

This study was undertaken to better understand the pathogenesis of squamous cell carcinoma (SCC) of the pancreas and to determine if inhibiting the p21-activated kinase 4 (PAK4) activity could be used for treating pancreatic SCCs. Here are the context of the research and preliminary results: Although significant research has been done on adenocarcinoma of the pancreas, the current understanding of pancreatic SCCs remains limited, and there is no known cure for it. We established a mouse model of pancreatic SCCs by over-expressing the transcription factor p63 in the pancreatic ductal epithelial-specific manner. Our working hypothesis was that dysregulation of p63-mediate transcriptional networks can lead to the development of pancreatic SCCs. As the stability of p63 is regulated at the protein level by phosphorylation, we searched for kinase that stabilizes p63 and identified PAK4 kinase. Although previous studies have shown that PAK4 is frequently overexpressed in ductal adenocarcinoma of the pancreas, the involvement of PAK4 in the pathogenesis of pancreatic SCCs remained unclear. We have demonstrated that suppression of the PAK4 kinase activity by a small molecule inhibitor reduced p63 levels in cellular models of pancreatic SCCs, leading to the complete suppression of their proliferation in vitro. These results indicate that PAK4 kinase can serve as a novel therapeutic target for some pancreatic SCCs and that PAK4-mediated stabilization/upregulation of the transcription factor p63 can be the key molecular event underlying the pathogenesis of pancreatic SCCs. We will use our mouse models in future studies to determine if administration of the PAK4 inhibitor in vivo can be used as an effective therapy for the treatment and prevention of the development of this most dangerous cancer subtype in human cancers.

Repeated exposure to fractional CO2 laser delays squamous cell carcinoma formation and prevents clinical and subclinical photodamage visualized by line-field confocal optical coherence tomography and histology.

Ablative fractional laser (AFL) is a well-established modality for treating ultraviolet radiation (UVR)-induced skin photodamage. We aimed to investigate the potential of AFL to delay squamous cell carcinoma(SCC) formation and prevent photodamage in a preclinical UVR-induced SCC model. Hairless C3.Cg-Hrhr /TifBomTac mice (n = 50) were exposed to UVR three timesweekly throughout the study. UV-exposed mice were randomized to two groups that received dorsal CO2 AFL (10 mJ/mb, 10% density) or no treatment. AFL was performed every other week for a total of 16 weeks (nine treatments in total). The primary outcome was time to tumor occurrence. In a subset of mice on Day 150, prevention of clinical photodamage was assessed by examination of skin tightness and dyspigmentation. Concomitantly, assessment of subclinical photoprevention based on normalization of keratinocyte dysplasia, dermo-fiber morphology (collagen and elastin fibers), and skin thickness, was performed using line-field confocal optical coherence tomography (LC-OCT) and histology. Repeated AFL treatments delayed SCC tumor development compared to UVR control mice by 12, 19, and 30 days for first, second, and third tumors, respectively (p ≤ 0.0017). Compared to UVR controls, AFL prevented photodamage both clinically and subclinically, based on LC-OCT and histology. In the epidermal layer, AFL imparted photopreventative effects including reduced dyspigmentation and keratinocyte dysplasia (1 vs. 2.5, p = 0.0079) and partial normalization of the epidermal thickness (p < 0.0001). In the dermis, AFL led to twofold greater skin tightness (p = 0.0079), improved dermo-fiber structure, and dermal thickness (p = 0.0011). In conclusion, repeated AFL treatments of UVR-exposed skin significantly delayed SCC tumor formation and prevented clinical and imaging-assessed subclinical signs of photodamage, indicating a potential for AFL in prevention strategies for SCC and photodamage in high-risk populations.

Chemopreventive Properties of Black Raspberries and Strawberries in Esophageal Cancer Review.

Esophageal cancer is one of the most fetal malignancies in the world. Esophageal squamous cell carcinoma (SCC) and esophageal adenocarcinoma (AC) are two main types of esophageal cancer and each with distinct epidemiological, etiological and histopathological characteristics. The continued global prevalence of tobacco use and alcohol consumption, coupled with limited intake of fresh fruits and vegetables, ensures that esophageal cancer will remain one of the major health threats. In addition to promoting quitting smoking and alcohol abuse, one of the strategies of cancer prevention is to identify foods, food components, or dietary patterns that can prevent or delay the onset of esophageal cancer. A food-based approach has the advantage of a complex of mixtures of bioactive components simultaneously targeting multiple processes in carcinogenesis. We have employed a preclinical rodent model of esophageal SCC to assess the effects of black raspberries (BRB) and strawberries. Our investigations demonstrate that BRB and strawberries are potent inhibitors of esophageal cancer. To prepare for this review, a literature search was performed to screen BRB and strawberries against esophageal cancer using electronic databases from PubMed, Science Direct and Google Scholar. Search was conducted covering the period from January 2000 to June 2022. Our present review has provided a systematic review about chemopreventive effects of BRB and strawberries in esophageal cancer by collecting and compiling diverse research findings from the above sources. In this review, we discussed the anti-tumor potentials of BRB and strawberries in esophageal SCC and esophageal AC separately. For each cancer type, we discuss animal models and research findings from both animal bioassays and human clinical studies. We also discuss the potential mechanisms of action of berries and their key bioactive components.

The Use of Panitumumab-IRDye800CW in a Novel Murine Model for Conjunctival Squamous Cell Carcinoma.

PurposeConjunctival squamous cell carcinoma (SCC) is a sight-threatening ocular surface malignancy with the primary treatment modality being surgical resection. To evaluate surgical imaging modalities to improve surgical resection, we established a novel murine model for conjunctival SCC to demonstrate the utility of panitumumab-IRDye800, a fluorescently labeled anti-epidermal growth factor receptor (EGFR) antibody.MethodsNOD-scid IL2Rgammanull (NSG) mice received subconjunctival injection of UM-SCC-1 or SCC-9, head and neck SCC cell lines. On tumor growth, mice were injected with Panitumumab-IRDye800CW, and imaged with a small animal imaging system and optical coherence tomography (OCT). Immunohistochemistry for SCC markers were used to confirm tumor origin.ResultsSeventy-five percent (N = 4) of the UM-SCC-1 group developed aggressive, rapidly growing tumors that were P40 and EGFR positive within two weeks of inoculation. The SCC-9 tumors failed to demonstrate any growth (N = 4). Ocular tumors demonstrated high fluorescence levels with a tumor to background ratio of 3.8.ConclusionsSubconjunctival injections are an appropriate technique to create in vivo models for assessing treatment modalities and novel therapies in conjunctival SCC.Translational RelevanceThis model demonstrates Panitumumab-IRDye800CW's utility in the ophthalmic setting and suggests that clinical trials may be warranted.

LB882 Dissecting mechanisms of responsiveness to the combination therapy of radiation and anti-PD-L1/anti-TGFb treatment in murine squamous cell carcinoma models

Squamous cell carcinoma (SCC) is often marked by an immunosuppressive tumor microenvironment, particularly in metastatic/recurrent disease. The purpose of this study is to evaluate the anti-tumor efficacy of and mechanisms of response to the combination therapy of radiotherapy (RT) and anti-PD-L1/anti-TGFb treatment using SCC models. We transplanted tumor cells derived from either carcinogen-induced SCCs or spontaneous SCCs of K15.Kras12D/Smad4-/- mice to syngeneic mouse recipients and subjected them to RT and anti-PD-L1/anti-TGFb therapy.

Abstract 6196: Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma

Abstract Fanconi anemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink (ICL) repair resulting in chromosome breakage. The FA repair pathway comprises at least 22 FANC proteins including BRCA1 and BRCA2 and protects against carcinogenic endogenous and exogenous aldehydes. Individuals with FA are hundreds to thousands-fold more likely to develop head and neck (HNSCC), esophageal and anogenital squamous cell carcinomas (SCCs) with a median onset age of 31 years. The aggressive nature of these tumors and poor patient tolerance of platinum and radiation-based therapy have been associated with short survival in FA. Molecular studies of SCCs from individuals with FA (FA SCCs) have been limited, and it is unclear how they relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or human papillomavirus (HPV) infection. Here, by sequencing FA SCCs, we demonstrate that the primary genomic signature of FA-deficiency is the presence of a high number of structural variants (SVs). SVs are enriched for small deletions, unbalanced translocations, and fold-back inversions that arise in the context of TP53 loss. The SV breakpoints preferentially localize to early replicating regions, common fragile sites, tandem repeats, and SINE elements. SVs are often connected forming complex rearrangements. Resultant genomic instability underlies elevated copy number alteration (CNA) rates of key HNSCC-associated genes, including PIK3CA, MYC, CSMD1, PTPRD, YAP1, MXD4, and EGFR. In contrast to sporadic HNSCC, we find no evidence of HPV infection in FA HNSCC, although positive cases were identified in gynecologic tumors. A murine allograft model of FA pathway-deficient SCC was enriched in SVs, exhibited dramatic tumor growth advantage, more rapid epithelial-to-mesenchymal transition, and enhanced autonomous inflammatory signaling when compared to an FA pathway-proficient model. In light of the protective role of the FA pathway against SV formation uncovered here, and recent findings of FA pathway insufficiency in the setting of increased formaldehyde load resulting in hematopoietic stem cell failure and carcinogenesis, we propose that high copy-number instability in sporadic HNSCC may result from functional overload of the FA pathway by endogenous and exogenous DNA crosslinking agents. Our work lays the foundation for improved FA patient treatment and demonstrates that FA SCC is a powerful model to study tumorigenesis resulting from DNA crosslinking damage. Citation Format: Andrew L. Webster, Mathijs A. Sanders, Krupa Patel, Ralf Dietrich, Raymond J. Noonan, Francis P. Lach, Ryan R. White, Audrey M. Goldfarb, Kevin Hadi, Matthew M. Edwards, Frank X. Donovan, Moonjung Jung, Sunandini Sridhar, Olivier Fedrigo, Huasong Tian, Joel Rosiene, Thomas Heineman, Jennifer Kennedy, Lorenzo Bean, Rasim O. Rosti, Rebecca Tryon, Ashlyn-Maree Gonzalez, Allana Rosenberg, Ji-Dung Luo, Thomas Carrol, Eunike Velleuer, Jeff C. Rastatter, Susanne I. Wells, Jordi Surrallés, Grover Bagby, Margaret L. MacMillan, John E. Wagner, Maria Cancio, Farid Boulad, Theresa Scognamiglio, Roger Vaughan, Amnon Koren, Marcin Imielinski, Settara Chandrasekharappa, Arleen D. Auerbach, Bhuvanesh Singh, David Kutler, Peter J. Campbell, Agata Smogorzewska. Fanconi anemia pathway deficiency drives copy number variation in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6196.

PHLDA3 Is an Important Downstream Mediator of p53 in Squamous Cell Carcinogenesis

Squamous cell carcinomas (SCCs) are one of the most frequent solid cancer types in humans and are derived from stratified epithelial cells found in various organs. SCCs derived from various organs share common important properties, including genomic abnormalities in the tumor suppressor gene p53. There is a carcinogen-induced mouse model of SCC that produces benign papilloma, some of which progress to advanced carcinoma and metastatic SCCs. These SCCs undergo key genetic alterations that are conserved between humans and mice, including alterations in the genomic p53 sequence, and are therefore an ideal system to study the mechanisms of SCC tumorigenesis. Using this SCC model, we show that the PHLDA3 gene, a p53-target gene encoding a protein kinase B repressor, is involved in the suppression of benign and metastatic tumor development. Loss of PHLDA3 induces an epithelial‒mesenchymal transition and can complement p53 loss in the formation of metastatic tumors. We also show that in human patients with SCC, low PHLDA3 expression is associated with a poorer prognosis. Collectively, this study identifies PHLDA3 as an important downstream molecule of p53 involved in SCC development and progression.

Characterization of Permeability Barrier Dysfunction in a Murine Model of Cutaneous Field Cancerization Following Chronic UV-B Irradiation: Implications for the Pathogenesis of Skin Cancer.

Simple SummaryIn the present work, we developed an experimental preclinical model of skin with cutaneous field cancerization after chronic UV-B light exposure in an immunologically intact mouse model (SKH1 aged mice). We observed impairments in the transepidermal water loss, stratum corneum hydration, and surface pH. We also detected a marked hyperkeratotic hyperplasia of the epidermis, induction of keratinocyte hyperproliferation, incidental actinic keratosis, and in situ squamous cell carcinomas in the UV-B light-irradiated groups. In this context, the association between the permeability barrier impairment and keratinocyte hyperproliferation might be considered a new target in the management of skin with cutaneous field cancerization. As current therapeutic approaches to actinic keratosis and cutaneous field cancerization only focus on the direct antineoplastic, immunomodulatory, or photodynamic effects of approved topical drugs, this mouse model of skin with cutaneous field cancerization might be helpful for both the identification and screening of potentially new preventive strategies or treatments (e.g., skin barrier therapies).Chronic ultraviolet B (UV-B) irradiation is known to be one of the most important hazards acting on the skin and poses a risk of developing photoaging, skin with cutaneous field cancerization (CFC), actinic keratosis (AKs), and squamous cell carcinomas (SCCs). Most of the UV-B light is absorbed in the epidermis, affecting the outermost cell layers, the stratum corneum, and the stratum granulosum, which protects against this radiation and tries to maintain the permeability barrier. In the present work, we show an impairment in the transepidermal water loss, stratum corneum hydration, and surface pH after chronic UV-B light exposure in an immunologically intact mouse model (SKH1 aged mice) of skin with CFC. Macroscopic lesions of AKs and SCCs may develop synchronically or over time on the same cutaneous surface due to both the presence of subclinical AKs and in situ SCC, but also the accumulation of different mutations in keratinocytes. Focusing on skin with CFC, yet without the pathological criteria of AKs or SCC, the presence of p53 immunopositive patches (PIPs) within the epidermis is associated with these UV-B-induced mutations. Reactive epidermis to chronic UV-B exposure correlated with a marked hyperkeratotic hyperplasia, hypergranulosis, and induction of keratinocyte hyperproliferation, while expressing an upregulation of filaggrin, loricrin, and involucrin immunostaining. However, incidental AKs and in situ SCC might show neither hypergranulosis nor upregulation of differentiation markers in the upper epidermis. Despite the overexpression of filaggrin, loricrin, involucrin, lipid enzymes, and ATP-binding cassette subfamily A member 12 (ABCA12) after chronic UV-B irradiation, the permeability barrier, stratum corneum hydration, and surface pH were severely compromised in the skin with CFC. We interpret these results as an attempt to restore the permeability barrier homeostasis by the reactive epidermis, which fails due to ultrastructural losses in stratum corneum integrity, higher pH on skin surface, abundant mast cells in the dermis, and the common presence of incidental AKs and in situ SCC. As far as we know, this is the first time that the permeability barrier has been studied in the skin with CFC in a murine model of SCC induced after chronic UV-B irradiation at high doses. The impairment in the permeability barrier and the consequent keratinocyte hyperproliferation in the skin of CFC might play a role in the physiopathology of AKs and SCCs.

Development of a prophylactic vaccine for lung squamous cell carcinoma

Abstract High-grade bronchial dysplasia is a marker for high risk of lung squamous cell carcinoma (SCC). Cancer vaccines targeting dysplasia could prevent the progression to SCC and decrease lung cancer incidence in population at risk. In order to develop a vaccine to prevent lung SCC we need to identify antigens and epitopes within them able to elicit a potent Type I anti-tumor immune response. One caveat to develop a prophylactic vaccine targeting dysplasia is that driver mutations (neo-antigens) are not known. Many mutations appear late in lung cancer and are not shared between patients. However, upregulated non-mutated proteins are needed from the early stages of cancer to support the new proliferation requirements of the cells, are common between patients and are good candidates for a preventive vaccine. Type I CD4+ T cells (Th1) secreting interferon-gamma (IFN-g) are necessary for enhanced function of antigen presenting cells, epitope spreading and activation of cytotoxic CD8+ T cells. However, non-mutated tumor self-antigens frequently induce Type II CD4+ T cells (Th2), which secrete cytokines that inhibit the function of Th1 and CD8+ T cells. To develop a prophylactic lung cancer vaccine we have: (1) searched in public datasets genes that are upregulated in bronchial dysplasia and maintain upregulation in SCC; (2) used web based algorithms for class II epitope prediction followed by functional ELISPOT screening to identified epitopes eliciting IFN-g (Th1) and/or IL-10 (Th2) responses; (3) selected only the Th1 epitopes (excluding the Th2 epitopes) to evaluated efficacy of the vaccines in a chemically induced rodent model of SCC. Preliminary data shows that we can reduce dysplasia frequency in vaccinated mice treated with the carcinogen.

Risk Prediction Model for Cutaneous SCC in Cardiac Allograft Recipients

Post-transplant patients are at increased risk for squamous cell carcinoma (SCC) of the skin, secondary to long-term immunosuppression. This study was undertaken to identify risk factors using the UNOS database population. The study population of 1766 patients derived from the UNOS database (2000-2015) was divided into two equal groups. One group was used for testing and the other for validation. Descriptive statistics were used to characterize the study cohort. Cox's proportional hazards regression was used to investigate the effect of different risk factors for SCC post-heart transplantation. Multivariate analysis was used to test the correlation between SCC and potential risk factors. Hazard ratio (OR) and the 95% confidence interval (CI) were calculated for each risk factor. Statistical significance was set at p< 0.05. Forward sequential feature selection was done to select significant risk factors for multivariate modeling. The multivariate model was used to predict the risk of SCC at 5, 8 and 10 years post-transplant. Analyses were performed using MATLAB software from The Math Works, Inc. Receiver Operating Curves (ROCs) were generated to derive Area Under the Curve (AUC) to assess the quality of the prediction model. Multivariate analysis showed that Black, Hispanic and other races as compared to whites had an OR of 0.07,0.35, 0.17 (p=0.0000). Recipient PRAs had an OR of 0.97 (p=<0.0001). Age >60 years had an OR of 2.5 (p=0.0000). Males had an OR of 2.05 (p=0.000). The ROC curves generated showed AUC at 5, 8 and 10 years post-transplant of 0.76, 0.76, 0.76 respectively in the testing set and 0.79, 0.77, 0.78 respectively in the validation set (figure 1). Male sex, white race, age >60 years higher PRAs are major risk factors for SCC. The risk prediction model generated for the first time for SCC in post-cardiac transplant patients has a robust c-statistic of >0.75. This is important for risk stratification as SCC is the most common skin cancer seen in the post-cardiac transplant population.

Orthotopic tongue squamous cell carcinoma (SCC) model exhibiting a different tumor-infiltrating T-cell status with margin-restricted CD8+ T cells and regulatory T cell-dominance, compared to skin SCC

The immunological, and especially T cell, status of the tumor microenvironment affects tumor development and the efficacy of cancer treatment. To devise suitable combination therapies based on the results of murine tumor models, a more realistic orthotopic model is required. In this study, we generated a murine model of tongue squamous cell carcinoma (SCC), in which the tumor–immune cell interactions were recapitulated, and examined tumor- and T-cell status compared to a skin-transplanted SCC model by multiplex immunofluorescence staining for epidermal growth factor receptor, CD31, CD8, CD4, and Foxp3. Administration of SCCVII cells did not induce undesirable tissue damage or inflammation. In tongue SCC, abundant T-cell infiltration was observed at the tumor margin, but not in the core. Tongue SCC predominantly showed CD8+ T or Foxp3+ regulatory T cell (Treg)-infiltration. In contrast, skin-transplanted SCC showed abundant infiltration of T cells in the whole tumor area, which was dominated by Tregs. An orthotopic tongue SCC model showed differences in tumor and T-cell status compared to the skin-transplanted SCC model. Our tongue SCC model may enhance understanding of tumor-host interactions and enable evaluation of therapeutic efficacy.

LncRNA CISAL Inhibits BRCA1 Transcription by Forming a Tertiary Structure at Its Promoter.

SummaryCisplatin-based neoadjuvant chemotherapy has been shown to improve survival in patients with squamous cell carcinoma (SCC), but clinical biomarkers to predict chemosensitivity remain elusive. Here, we show the long noncoding RNA (lncRNA) LINC01011, which we termed cisplatin-sensitivity-associated lncRNA (CISAL), controls mitochondrial fission and cisplatin sensitivity by inhibiting BRCA1 transcription in tongue SCC (TSCC) models. Mechanistically, we found CISAL directly binds the BRCA1 promoter and forms an RNA-DNA triplex structure, sequestering BRCA1 transcription factor-GABPA away from the downstream regulatory binding region. Importantly, the clinical relevance of these findings is suggested by the significant association of CISAL and BRCA1 expression levels in TSCC tumors with neoadjuvant chemosensitivity and overall survival. We propose a new model where lncRNAs are tethered at gene promoter by RNA-DNA triplex formation, spatially sequestering transcription factors away from DNA-binding sites. Our study uncovers the potential of CISAL-BRCA1 signaling as a potential target to predict or improve chemosensitivity.

ADAP1 promotes invasive squamous cell carcinoma progression and predicts patient survival.

Invasive squamous cell carcinoma (SCC) is aggressive cancer with a high risk of recurrence and metastasis, but the critical determinants of its progression remain elusive. Here, we identify ADAP1, a GTPase-activating protein (GAP) for ARF6 up-regulated in TGF-β-responding invasive tumor cells, as a strong predictor of poor survival in early-stage SCC patients. Using a mouse model of SCC, we show that ADAP1 overexpression promotes invasive tumor progression by facilitating cell migration and breakdown of the basement membrane. We found that ADAP1-rich, TGF-β-responding tumor cells exhibit cytoplasmic laminin localization, which correlated with the absence of laminin and type IV collagen from the pericellular basement membrane. Interestingly, although tumors overexpressing a GAP activity-deficient mutant of ADAP1 resulted in morphologically complex tumors, those tumor cells failed to breach the basement membrane. Moreover, Adap1 deletion in tumor cells ameliorated the basement membrane breakdown and had less invading cells in the stroma. Our study demonstrates that ADAP1 is a critical mediator of TGF-β-induced cancer invasion and might be exploited for the treatment of high-risk SCC.

Dysregulated Tgfbr2/ERK-Smad4/SOX2 Signaling Promotes Lung Squamous Cell Carcinoma Formation.

Lung squamous cell carcinoma (SCC) is a common type of lung cancer. There is limited information on the genes and pathways that initiate lung SCC. Here, we report that loss of TGFβ type II receptor (Tgfbr2), frequently deleted in human lung cancer, led to predominant lung SCC development in KrasG12D mice with a short latency, high penetrance, and extensive metastases. Tgfbr2-loss-driven lung SCCs resembled the salient features of human lung SCC, including histopathology, inflammatory microenvironment, and biomarker expression. Surprisingly, loss of Smad4, a key mediator of Tgfbr2, failed to drive lung SCC; instead, low levels of phosphorylated ERK1/2, a Smad-independent downstream effector of Tgfbr2, were tightly associated with lung SCC in both mouse and human. Mechanistically, inhibition of phosphorylated ERK1/2 significantly upregulated the expression of SOX2, an oncogenic driver of lung SCC, and cooperated with SMAD4 repression to elevate SOX2. Inhibition of ERK1/2 in Smad4fl/fl ;KrasG12D mice led to extensive lung SCC formation that resembled the SCC phenotype of Tgfbr2-deficient mice. Overall, we reveal a key role of ERK1/2 in suppressing SCC formation and demonstrate that dysregulated Tgfbr2/ERK-Smad4/SOX2 signaling drives lung SCC formation. We also present a mouse model of metastatic lung SCC that may be valuable for screening therapeutic targets. SIGNIFICANCE: This study sheds new light on the mechanisms underlying lung SCC formation driven by mutated Kras.

Abstract 5088: Silibinin inhibits UVB-induced promotion and progression of microscopic basal cell carcinoma formation via targeting bone morphogenic protein 2

Abstract Non-melanoma skin cancers (NMSCs) account for about half of all malignancies diagnosed annually in the United States. Around 80% of NMSCs are basal cell carcinoma (BCC) and 20% are squamous cell carcinoma (SCC). Whereas the efficacy of several chemopreventive agents has been examined and reported against both BCC and SCC, a majority of these studies have focused on the test agent’s activity in a long-term setting to determine the amount of tumors formed. Notably, the studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of UVB-induced BCC formation, we excised skin samples from UVB exposed mice prior to tumor formation to study the promotion/progression of BCC and to determine the target/s of silibinin, a well-known skin cancer (SCC) chemopreventive agent, in tumor growth inhibition. Ptch+/- and Ptch+/+ mice were irradiated with UVB 240mJ/cm2 3 times per week with or without topically applied silibinin 5 times per week. As early as one month, we found that UVB exposure significantly increased the number of mast cells in Ptch+/- mice by about 48% (P&amp;lt;0.05), which was completely inhibited (to control levels) by silibinin topical treatments. In Ptch+/+ mice, which do not develop BCC tumors, we did not observe any increase in mast cells following UVB exposure, suggesting this could be a specific pathway in the development of BCC. To decipher the molecular mechanism of these findings, we performed a PCR profiler array analysis of several genes involved in signal transduction pathways which showed strong differences between Ptch+/+ and Ptch+/- mice that were unexposed, UVB irradiated, and silibinin treated. Most notably, following UVB exposure for 1 month, in Ptch+/- mice the expression of Bone morphogenetic protein 2 (BMP-2), Hairy/enhancer-of-split related with YRPW motif 1 (Hey1), and Inhibitor of DNA binding 1 (Id1) was significantly upregulated when compared to Ptch+/+ mice. Additional studies focusing on BMP-2 found that silibinin strongly inhibits UVB-induced expression of BMP-2 in Ptch+/- mouse skin. Consistent with these results, we also found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo. Regarding BCC formation, silibinin treatment inhibited UVB-induced microscopic BCC formation in Ptch+/- mice; microscopic tumor number and size were reduced by 73% and 84%, respectively. Together, our results suggest a possible role of BMP-2 in early stages of BCC development and that silibinin plausibly acts through BMP-2 to inhibit microscopic BCC formation. In conclusion, our previous studies in SCC models and current findings in BCC model suggest that silibinin could be a multi-target agent capable of being a chemopreventive agent for both types of NMSCs. Citation Format: Cynthia M. Rigby, Gagan Deep, Anil K. Jain, David J. Orlicky, Chapla Agarwal, Komal Raina, Rajesh Agarwal. Silibinin inhibits UVB-induced promotion and progression of microscopic basal cell carcinoma formation via targeting bone morphogenic protein 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5088.