cSCC Cell Lines Research Articles

Oncolytic activity of a coxsackievirus B3 strain in patient-derived cervical squamous cell carcinoma organoids and synergistic effect with paclitaxel

BackgroundCervical squamous cell carcinoma (CSCC) is a prevalent gynecological malignancy worldwide. Current treatments for CSCC can impact fertility and cause long-term complications, underscoring the need for new therapeutic strategies. Oncolytic virotherapy has emerged as a promising option for cancer treatment. Previous research has demonstrated the oncolytic activity of the coxsackievirus B3 strain 2035 A (CVB3/2035A) against various tumor types. This study aims to evaluate the clinical viability of CVB3/2035A for CSCC treatment, focusing on its oncolytic effect in patient-derived CSCC organoids.MethodsThe oncolytic effects of CVB3/2035A were investigated using human CSCC cell lines in vitro and mouse xenograft models in vivo. Preliminary tests for tumor-selectivity were conducted on patient-derived CSCC tissue samples and compared to normal cervical tissues ex vivo. Three patient-derived CSCC organoid lines were developed and treated with CVB3/2035A alone and in combination with paclitaxel. Both cytotoxicity and virus replication were evaluated in vitro.ResultsCVB3/2035A exhibited significant cytotoxic effects in human CSCC cell lines and xenograft mouse models. The virus selectively induced oncolysis in patient-derived CSCC tissue samples while sparing normal cervical tissues ex vivo. In patient-derived CSCC organoids, which retained the immunohistological characteristics of the original tumors, CVB3/2035A also demonstrated significant cytotoxic effects and efficient replication, as evidenced by increased viral titers and presence of viral nucleic acids and proteins. Notably, the combination of CVB3/2035A and paclitaxel resulted in enhanced cytotoxicity and viral replication.ConclusionsCVB3/2035A showed oncolytic activity in CSCC cell lines, xenografts, and patient-derived tissue cultures and organoids. Furthermore, the virus exhibited synergistic anti-tumor effects with paclitaxel against CSCC. These results suggest CVB3/2035A could serve as an alternative or adjunct to current CSCC chemotherapy regimens.

Clustering of RNA co-expression network identifies novel long non-coding RNA biomarkers in squamous cell carcinoma

Long non-coding RNAs (lncRNAs) have emerged as important players in cancer progression. Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer with increasing incidence worldwide. The prognosis of the metastatic cSCC is poor, and currently there are no established biomarkers to predict metastasis risk or specific therapeutic targets for advanced or metastatic cSCC. To elucidate the role of lncRNAs in cSCC, RNA sequencing of patient derived cSCC cell lines and normal human epidermal keratinocytes was performed. The correlation analysis of differentially expressed lncRNAs and protein-coding genes revealed six distinct gene clusters with one of the upregulated clusters featuring genes associated with cell motility. Upregulation of the expression of lncRNAs linked to cSCC cell motility in cSCC and head and neck SCC (HNSCC) cells was confirmed using qRT-PCR. Elevated expression of HOTTIP and LINC00543 was also noted in SCC tumors in vivo and was associated with poorer prognosis in HNSCC and lung SCC cohorts within TCGA data, respectively. Altogether, these findings uncover a novel set of lncRNAs implicated in cSCC cell locomotion. These lncRNAs may serve as potential novel biomarkers and as putative therapeutic targets for locally advanced and metastatic cSCC.

C1s targeting antibodies inhibit the growth of cutaneous squamous carcinoma cells

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. The incidence of cSCC is increasing globally and the prognosis of metastatic disease is poor. Currently there are no specific targeted therapies for advanced or metastatic cSCC. We have previously shown abundant expression of the complement classical pathway C1 complex components, serine proteases C1r and C1s in tumor cells in invasive cSCCs in vivo, whereas the expression of C1r and C1s was lower in cSCCs in situ, actinic keratoses and in normal skin. We have also shown that knockdown of C1s expression results in decreased viability and growth of cSCC cells by promoting apoptosis both in culture and in vivo. Here, we have studied the effect of specific IgG2a mouse monoclonal antibodies TNT003 and TNT005 targeting human C1s in five primary non-metastatic and three metastatic cSCC cell lines that show intracellular expression of C1s and secretion of C1s into the cell culture media. Treatment of cSCC cells with TNT003 and TNT005 significantly inhibited their growth and viability and promoted apoptosis of cSCC cells. These data indicate that TNT003 and TNT005 inhibit cSCC cell growth in culture and warrant further investigation of C1s targeted inhibition in additional in vitro and in vivo models of cSCC.

Echinatin suppresses cutaneous squamous cell carcinoma by targeting GSTM3-mediated ferroptosis

BackgroundCutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers for which effective drugs are urgently needed. Echinatin, a natural compound extracted from Glycyrrhiza plants, has shown promising antitumour effects. However, the efficacy and the direct target of echinatin in cSCC remain unclear. PurposeThis study conducted a systematic investigation of the antitumour effects of echinatin on cSCC and the underlying mechanisms involved. Study design and methodsThree cSCC cell lines, a xenograft model, and a UV-induced cSCC mouse model were used to investigate the potential protective effects of echinatin. The interactions between echinatin and glutathione S-transferase mu3 (GSTM3) and between echinatin and peroxiredoxin-2 (PRDX2) were evaluated by a proteome microarray assay, pull-down LC‒MS/MS analysis, surface plasmon resonance, and molecular docking. The potential mechanisms of GSTM3-mediated echinatin activity were analysed by using western blotting, lentivirus infection and small interfering RNA (siRNA) transfection. ResultsIn this study, we found that echinatin inhibited the proliferation and migration of cSCC cells but had no cytotoxic effect on primary human keratinocytes. Furthermore, echinatin significantly inhibited tumour growth in vivo. Mechanistically, our data showed that echinatin could directly bind to GSTM3 and PRDX2. Notably, echinatin inhibited GSTM3 and PRDX2 levels by promoting their proteasomal degradation, which led to the disruption of ROS production. We then revealed that echinatin increased mitochondrial ROS production by inhibiting GSTM3. Moreover, echinatin triggered ferroptosis by inhibiting GSTM3-mediated ferroptosis negative regulation (FNR) proteins. In addition, echinatin regulated GSTM3-mediated ROS/MAPK signalling. ConclusionEchinatin has good antitumour effects both in vitro and in vivo. Moreover, our findings indicate that GSTM3 and PRDX2 could function as viable targets of echinatin in cSCC. Consequently, echinatin represents a novel treatment for cSCC through the targeting of GSTM3-mediated ferroptosis.

P37 Drug repurposing for treatment of recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma

Abstract Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited skin disorder that leads to severe wounding and blistering of the skin. Patients with RDEB develop aggressive cutaneous squamous cell carcinoma (cSCC) with a mortality rate of approximately 90% by age 55 years. The current primary treatment is wide excision of tumours, often leading to amputations. Identifying new therapeutic avenues for RDEB cSCC remains a priority. Here, we approach this in two ways. Firstly, we identified a SMAD4-dependent sphingosine pathway response to endogenous transforming growth factor (TGF)-β, essential for RDEB cSCC proliferation which could be targeted as a potential therapeutic strategy. Secondly, we utilized a US Food and Drug Administration (FDA) screen of > 3000 drugs to identify potential druggable pathways that could be used to treat RDEB cSCC. Methods RDEB cSCC cell lines were used to test Siponimod, a sphingosine-1 receptor modulator, on proliferation, clonogenicity and invasion in vitro. Siponimod was used in vivo to test tumour growth effect of targeting the sphingosine pathway. We carried out an FDA-approved drug screen on four RDEB cSCC cell lines using the Opera Phenix high-throughput microscope to identify targets. Results Treatment of RDEB cSCC cell lines with Siponimod showed compromised proliferation compared with untreated cells in addition to significant reduction in colony formation. There was a significant decrease in the invasive capability of RDEB SCC cells. Treatment of normal human keratinocytes showed no effect on proliferation. Our FDA-approved screen identified multiple drugs that target MEK, mechanistic target of rapamycin complex, histone deacetylase, signal transducer and activator of transcription, and topoisomerase I/II in the top 50 hits. The most common target was DNA damage/repair pathways, but cytoskeletal signalling, transmembrane transporters and proteases were identified as other potential avenues for treatment of RDEB cSCC. Conclusions There is an unmet need for therapeutic options in RDEB cSCC. Here, we have demonstrated the potential of repurposing previously licensed drugs to identify treatment avenues for RDEB cSCC.

Transglutaminase 3 regulates cutaneous squamous carcinoma differentiation and inhibits progression via PI3K-AKT signaling pathway-mediated Keratin 14 degradation.

Cutaneous squamous carcinoma is the second most common epithelial malignancy, associated with significant morbidity, mortality, and economic burden. However, the mechanisms underlying cSCC remain poorly understood. In this study, we identified TGM3 as a novel cSCC tumor suppressor that acts via the PI3K-AKT axis. RT-qPCR, IHC and western blotting were employed to assess TGM3 levels. TGM3-overexpression/knockdown cSCC cell lines were utilized to detect TGM3's impact on epithelial differentiation as well as tumor cell proliferation, migration, and invasion in vitro. Additionally, subcutaneous xenograft tumor models were employed to examine the effect of TGM3 knockdown on tumor growth in vivo. Finally, molecular and biochemical approaches were employed to gain insight into the tumor-suppressing mechanisms of TGM3. TGM3 expression was increased in well-differentiated cSCC tumors, whereas it was decreased in poor-differentiated cSCC tumors. Loss of TGM3 is associated with poor differentiation and a high recurrence rate in patients with cSCC. TGM3 exhibited tumor-suppressing activity by regulating cell proliferation, migration, and invasion both in vitro and in vivo. As a novel cSCC tumor differentiation marker, TGM3 expression was positively correlated with cell differentiation. In addition, our results demonstrated an interaction between TGM3 and KRT14 that aids in the degradation of KRT14. TGM3 deficiency disrupts keratinocytes differentiation, and ultimately leads to tumorigenesis. Furthermore, RNA-sequence analysis revealed that loss of TGM3 enhanced EMT via the PI3K-AKT signaling pathway. Deguelin, a PI3K-AKT inhibitor, blocked cSCC tumor growth induced by TGM3 knockdown in vivo. Taken together, TGM3 inhibits cSCC tumor growth via PI3K-AKT signaling, which could also serve as a tumor differentiation marker and a potential therapeutic target for cSCC. Proposed model depicted the mechanism by which TGM3 suppress cSCC development. TGM3 reduces the phosphorylation level of AKT and degrades KRT14. In the epithelial cell layer, TGM3 exhibits a characteristic pattern of increasing expression from bottom to top, while KRT14 and pAKT are the opposite. Loss of TGM3 leads to reduced degradation of KRT14 and activation of pAKT, disrupting keratinocyte differentiation, and eventually resulting in the occurrence of low-differentiated cSCC.

Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

Application of Doxorubicin-loaded PLGA nanoparticles targeting both tumor cells and cancer-associated fibroblasts on 3D human skin equivalents mimicking melanoma and cutaneous squamous cell carcinoma

Nanoparticle (NP) use in cancer therapy is extensively studied in skin cancers. Cancer-associated fibroblasts (CAFs), a major tumor microenvironment (TME) component, promote cancer progression, making dual targeting of cancer cells and CAFs an effective therapy. However, dual NP-based targeting therapy on both tumor cells and CAFs is poorly investigated in skin cancers. Herein, we prepared and characterized doxorubicin-loaded PLGA NPs (DOX@PLGA NPs) and studied their anti-tumor effects on cutaneous melanoma (SKCM)(AN, M14) and cutaneous squamous cell carcinoma (cSCC) (MET1, MET2) cell lines in monolayer, as well as their impact on CAF deactivation. Then, we established 3D full thickness models (FTM) models of SKCM and cSCC using AN or MET2 cells on dermis matrix populated with CAFs respectively, and assessed the NPs' tumor penetration, tumor-killing ability, and CAF phenotype regulation through both topical administration and intradermal injection. The results show that, in monolayer, DOX@PLGA NPs inhibited cancer cell growth and induced apoptosis in a dose- and time-dependent manner, with a weaker effect on CAFs. DOX@PLGA NPs reduced CAF-marker expression and had successful anti-tumor effects in 3D skin cancer FTMs, with decreased tumor-load and invasion. DOX@PLGA NPs also showed great delivery potential in the FTMs and could be used as a platform for future functional study of NPs in skin cancers using human-derived skin equivalents. This study provides promising evidence for the potential of DOX@PLGA NPs in dual targeting therapy for SKCM and cSCC.

Knockdown of Simulated-Solar-Radiation-Sensitive miR-205-5p Does Not Induce Progression of Cutaneous Squamous Cell Carcinoma In Vitro.

Solar radiation is the main risk factor for cSCC development, yet it is unclear whether the progression of cSCC is promoted by solar radiation in the same way as initial tumorigenesis. Additionally, the role of miRNAs, which exert crucial functions in various tumors, needs to be further elucidated in the context of cSCC progression and connection to solar radiation. Thus, we chronically irradiated five cSCC cell lines (Met-1, Met-4, SCC-12, SCC-13, SCL-II) with a custom-built irradiation device mimicking the solar spectrum (UVB, UVA, visible light (VIS), and near-infrared (IRA)). Subsequently, miRNA expression of 51 cancer-associated miRNAs was scrutinized using a flow cytometric multiplex quantification assay (FirePlex®, Abcam). In total, nine miRNAs were differentially expressed in cell-type-specific as well as universal manners. miR-205-5p was the only miRNA downregulated after SSR-irradiation in agreement with previously gathered data in tissue samples. However, inhibition of miR-205-5p with an antagomir did not affect cell cycle, cell growth, apoptosis, or migration in vitro despite transient upregulation of oncogenic target genes after miR-205-5p knockdown. These results render miR-205-5p an unlikely intracellular effector in cSCC progression. Thus, effects on intercellular communication in cSCC or the simultaneous examination of complementary miRNA sets should be investigated.

Modified 5-aminolevulinic acid photodynamic therapy induces cutaneous squamous cell carcinoma cell pyroptosis via the JNK signaling pathway

Modified 5-aminolevulinic acid photodynamic therapy (M-PDT) is a novel therapeutic modality for cutaneous squamous cell carcinoma (cSCC) that is reported to be effective and well tolerated. However, the mechanisms underlying its antitumor effects are not fully understood. In this research, we investigated the effects of M-PDT on pyroptosis, a form of programmed cell death characterized by cell swelling, ruptures of cell membrane, and inflammatory cytokine release, in two human cSCC cell lines, SCL-1 and HSC-5. We found that M-PDT triggered pyroptosis in a dose-dependent manner, as evidenced by increased lactate dehydrogenase release, propidium iodide staining, and expression of pyroptosis-related proteins, such as NLR family pyrin domain containing 3 (NLRP3), N-terminal of gasdermin D (N-GSDMD), cleaved caspase-1, and mature interleukin 1 beta (IL-1B) in both cell lines. This process was inhibited by treatment with MCC950, an NLRP3-specific inhibitor, suggesting the involvement of the NLRP3 inflammasome in M-PDT-induced pyroptosis. We also demonstrated that M-PDT activated c-Jun N-terminal kinase (JNK) signaling, which is required for pyroptosis induction, as treatment with SP600125, a JNK inhibitor, suppressed the expression of pyroptosis-related proteins after M-PDT. JNK activation enhanced M-PDT-induced pyroptosis, highlighting the significance of the JNK pathway in M-PDT. Moreover, M-PDT increased intracellular reactive oxygen species (ROS) levels, which are responsible for JNK activation and pyroptosis induction. In summary, our results revealed that M-PDT triggers pyroptosis through ROS-mediated JNK activation and subsequent NLRP3 inflammasome activation in cSCC cells, providing a better understanding of the molecular mechanism of M-PDT and promoting its clinical application.

P21 Tyrosine and serine STAT3 phosphorylation drives cutaneous squamous cell carcinoma in recessive dystrophic epidermolysis bullosa as demonstrated using a 3D in vitro model

Abstract The early onset of aggressive cutaneous squamous cell carcinoma (cSCC) and its rapid progression in recessive dystrophic epidermolysis bullosa (RDEB) leads to nearly 100% mortality rates in these patients. Elevated levels of phospho-STAT3 on tyrosine residue (STAT3Tyr705) have been intensively demonstrated in RDEB-derived cSCC cells, demonstrating that the constitutive activation and dysregulation of the Janus kinase (JAK)/STAT pathway plays a role in RDEB cSCC pathogenesis. We efficiently applied ruxolitinib (JAK1/2 inhibitor that blocks phosphorylation on STAT3Tyr705) to a RDEB–cSCC xenograph mouse model, reducing the size of small tumours but found that it was insufficient to reduce bigger tumours completely. Recently, phosphorylation at serine-727 (STAT3Ser727) has been shown to be relevant in the evolution of some cancers, but it has not yet been studied in RDEB cSCC. We hypothesized that phosphorylation at serine-727 of the STAT3 pathway might play a role in RDEB cSCC tumour development and progression, and could not be inhibited by ruxolitinib. To validate our hypothesis, we studied two RDEB cSCC human patient samples with different degrees of dedifferentiation (normal RDEB skin, edge of the tumour and centre of the RDEB cSCC). We demonstrated that STAT3Tyr705 was preferentially upregulated in the normal RDEB skin followed by the tumour edge skin, presenting less phosphorylation on the tumour. When we analysed STAT3 phosphorylation on STAT3Ser727, we could see that it presented the opposite phosphorylation pattern, being more phosphorylated in the tumour and less in normal RDEB skin. Furthermore, we validated the STAT3Ser727residue pattern across other cSCC cell lines and found that serine phosphorylation is present in other SCCs, demonstrating its crucial role in tumour development. To further investigate and analyse cancer progression in more detail, an easy and robust RDEB cSCC model is instrumental. Therefore, we generated RDEB cSCC spheroids in vitro, which has four key advantages over the previous models developed: (i) it is easy to generate; (ii) small amounts of cells are required; (iii) it is suitable for drug testing; and (iv) it can be used to recapitulate different stages of tumour development. We have been able to control their size and use them for drug testing, observing a 20% size decrease after 6 days of ruxolitinib treatment. Taken together, this three-dimensional RDEB cSCC in vitro model, along with analysis of human samples, will allow us to better understand RDEB cSCC progression at different stages, allowing us to investigate new treatments for RDEB cSCC.

Targeting Exportin 1 as treatment for cutaneous squamous cell carcinoma.

e21564 Background: Cutaneous squamous cell carcinomas (cSCC) are among the most frequent solid cancers in humans and in the U.S., cSCC cases are increasing. Molecular mechanisms transitioning premalignant lesions to cancer are not well characterized. As a result, targeted therapies need to be identified to treat these lesions. Preliminary data shows that Exportin 1 (XPO1) is one of the major proteins regulating nuclear export of proteins, helping to maintain cellular homeostasis. Previous work showed that Exportin 1 modulates the nuclear:cytoplasmic ratio of signaling proteins in skin cancer; resulting in an anti-apoptotic effect by adjusting the nuclear-cytoplasmic balance of pro-survival proteins such as CDC25A and Survivin. XPO1 expression is upregulated in many cancers, thereby affecting the nuclear:cytoplasmic ratio of many pro-survival proteins and enhancing tumorigenesis. This research project was designed to investigate the effects of XPO1 inhibition on cSCC survival. Selinexor is FDA cleared for treatment of multiple myeloma patients. Selinexor is a selective inhibitor of XPO1 regulated nuclear transport causing target molecules to remain within the cell leading to decreased cancer cell growth and decreased cancer cell survival. We hypothesize that inhibiting XPO1 with Selinexor or by CRISPRi genetic knockdown of XPO1 in cSCC cells will reduce proliferation and increase apoptosis in cSCC cells. Methods: Human cSCC cell lines SCC13 and SRB12 were seeded in 96-well plates at 8,000 cells/well density and 3,000 cells/well, respectively. Cells were treated with varying concentrations of Selinexor (0.005mM – 5mM) or DMSO as vehicle control. At 24-hour intervals, cell imaging and Neutral red staining assays were conducted to determine the IC50 concentration for Selinexor in cSCC cell lines. SCC13 cells stably expressing dCas9-KRAB and a doxycycline-inducible XPO1 guide RNA were used for CRISPRi experiments. Cells were treated with doxycycline daily and apoptosis was assayed by Caspase Glo 3/7 daily for four days. CRIPSRi enabled a doxycycline inducible XPO1 knock down on SCC13 cells allowing us to disrupt the cellular transporter protein XPO1. Results: Selinexor treatment of SRB12 cells resulted in an IC50 of 1.174 μM and 0.071 μM for SCC13. In contrast, normal human keratinocyte cell lines had higher IC50 values of 2.18 μM for HaCaT and 5.29 μM for KerCT cells. Apoptosis significantly increased after 72 and 96 hours of XPO1 knock down in SCC13 cells measured by Caspase Glo. Conclusions: These results document how XPO1 inhibition reduces proliferation and is toxic to both normal keratinocytes and cSCC cells. Additionally, cSCC cells had lower IC50 values for Selinexor, suggesting cSCC may be more sensitive to XPO1 inhibition compared to normal keratinocytes. Further, targeting XPO1 also increases apoptosis in these cells. This data supports the hypothesis that targeting XPO1 may be effective in treating cSCC.

Eribulin inhibits growth of cutaneous squamous cell carcinoma cell lines and a novel patient-derived xenograft

Advanced cutaneous squamous cell carcinoma (cSCC) is treated with chemotherapy and/or radiotherapy, but these typically fail to achieve satisfactory clinical outcomes. There have been no preclinical studies to evaluate the effectiveness of eribulin against cSCC. Here, we examine the effects of eribulin using cSCC cell lines and a novel cSCC patient-derived xenograft (PDX) model. In the cSCC cell lines (A431 and DJM-1 cells), eribulin was found to inhibit tumor cell proliferation in vitro as assessed by cell ATP levels. DNA content analysis by fluorescence-activated cell sorting (FACS) showed that eribulin induced G2/M cell cycle arrest and apoptosis. In xenograft models of cSCC cell lines, the administration of eribulin suppressed tumor growth in vivo. We also developed a cSCC patient-derived xenograft (PDX) which reproduces the histological and genetic characteristics of a primary tumor. Pathogenic mutations in TP53 and ARID2 were detected in the patient’s metastatic tumor and in the PDX tumor. The cSCC-PDX responded well to the administration of eribulin and cisplatin. In conclusion, the present study shows the promising antineoplastic effects of eribulin in cSCC. Also, we established a novel cSCC-PDX model that preserves the patient’s tumor. This PDX could assist researchers who are exploring innovative therapies for cSCC.

Abstract 4448: Chemokine (C-X-C motif) ligand 17 promotes cutaneous squamous cell carcinoma via modulating tumor-immune evasion

Abstract Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer, striking 200,000 Americans annually. Further, organ transplant patients are at significantly increased risk of cSCC compared to the general population. Excision of cSCC of the head and neck area results in significant facial disfigurement. An increased understanding of the mechanisms involved in the pathogenesis of cSCC could identify means to prevent, inhibit, and reverse this process. Chemokine (C-X-C motif) ligand 17 (CXCL17) is the latest chemokine family member, and an increased CXCL17 protein expression was observed in both mouse and human cSCC cell lines. Further, deletion of CXCL17 was associated with significant inhibition of tumor cell-intrinsic properties such as proliferation, migration, and motility. CXCL17 is identified as a potent chemoattractant for macrophages, dendritic cells and myeloid-derived suppressive cells. Accordingly, utilizing a syngeneic, tumor-cell xenograft cSCC mouse model, we evaluated the effect of CXCL17 deletion on cSCC tumor-immune evasion and elucidated the underlying mechanism. Deletion of CXCL17 was associated with a significant reduction in tumor volume compared to the wild-type counterparts. Further, CXCL17 deleted cSCC tumor cell xenografts exhibited a significant increase in CD8+, cytotoxic T cells in the tumor microenvironment, suggesting an important role of CXCL17 in mediating tumor-immune evasion. Interestingly, treatment with CXCL17 induced macrophage M2 polarization and promoted macrophage efferocytosis via modulating efferocytotic machinery proteins such as MERTK, TIM4, GAS6 and AXL. Our studies have established substantial evidence for the role of CXCL17 in modulating tumor-cell extrinsic properties to affect the progression of cSCC. Citation Format: Alok R. Khandelwal, Rema Anisha Kandula, Emily Daniel, Md Maksudul Alam, Henry Craighead, Tara Moore-Medlin, Cherie-Ann O. Nathan. Chemokine (C-X-C motif) ligand 17 promotes cutaneous squamous cell carcinoma via modulating tumor-immune evasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4448.

The long non-coding RNA PVT1 promotes tumorigenesis of cutaneous squamous cell carcinoma via interaction with 4EBP1

The long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) plays an oncogenic role in multiple cancers due to its high expression. However, the expression and associated regulatory mechanisms of PVT1 in cutaneous squamous cell carcinoma (cSCC) remain unclear. Our results revealed that PVT1 was highly upregulated in cSCC tissues and cSCC cell lines. To determine the functional role of PVT1 in cSCC, we constructed a stable knockdown cell model of PVT1 in the A431 and COLO16 cell lines using a lentiviral approach. Xenograft tumor experiments of nude mice in vivo, and colony formation, CCK-8, and EdU assays in vitro demonstrated that knockdown of PVT1 could widely suppress cell proliferation in vivo and in vitro. In addition, PVT1 knockdown induced cell cycle arrest and promoted apoptosis, as detected by flow cytometry analysis. Wound healing and transwell assays revealed that PVT1 knockdown significantly inhibited the migration and invasion of CSCC cell lines. To gain insight into the tumorigenic mechanism and explore the potential target molecules of PVT1, we employed label-free quantitative proteomic analysis. The GO, KEGG enrichment, and protein–protein interaction (PPI) networks suggested that 4E-binding protein 1 (4EBP1) is the possible downstream target effector of PVT1, which was validated by western blot analysis. PVT1 silencing markedly decreased 4EBP1 protein expression levels and directly bound 4EBP1 in the cytoplasm of cSCC cells. 4EBP1 overexpression counteracted the effects of PVT1 knockdown on tumorigenesis in cSCC cells, including cell proliferation, apoptosis, migration, and invasion. Our findings provide strong evidence that PVT1 is an oncogene which plays a role in tumorigenesis of cSCC, that PVT1 may interact with 4EBP1 in the cytoplasm as an underlying mechanism in cSCC carcinogenesis, and that PVT1 combined with 4EBP1 may serve as a potential new therapeutic target for cSCC.

Proline-rich 11 (PRR11) promotes the progression of cutaneous squamous cell carcinoma by activating the EGFR signaling pathway.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin malignancies, and its incidence rate is increasing worldwide. Proline-rich 11 (PRR11) has been reported to be involved in the occurrence and development of various tumors. However, the role of PRR11 in cSCC remains unknown. In the present study, we observed upregulated expression of PRR11 in cSCC tissues and cell lines. Knockdown of PRR11 in the cSCC cell lines A431 and SCL-1 inhibited cell proliferation by inducing cell cycle arrest during the G1/S phase transition, promoted cell apoptosis, and reduced cell migration and invasion in vitro. Conversely, overexpression of PRR11 promoted cell proliferation, decreased cell apoptosis, and enhanced cell migration and invasion. PRR11 knockdown also inhibited cSCC tumor growth in a mouse xenograft model. Mechanistic investigations by RNA sequencing revealed that 891 genes were differentially expressed genes between cells with PRR11 knockdown and control cells. Enrichment analysis of different genes showed that the epidermal growth factor receptor (EGFR) signaling pathway was the top enriched pathway. We further validated that PRR11 induced EGFR pathway activity, which contributed to cSCC progression. These data suggest that PRR11 may serve as a novel therapeutic target in cSCC.

NPTX2 Promotes Epithelial–Mesenchymal Transition in Cutaneous Squamous Cell Carcinoma through METTL3-Mediated N6-Methyladenosine Methylation of SNAIL

Cutaneous squamous cell carcinoma (cSCC) is the second most common type of skin cancer. NPTX2, a member of the neuronal pentraxin family, is reported to play inconsistent roles in different cancers. The role and mechanism of NPTX2 in cSCC remain unclear. In this study, we found that NPTX2 was overexpressed in both skin lesions and cell lines of cSCC. Invitro studies showed that NPTX2 facilitated cell proliferation, migration, invasion, colony formation, and epithelial‒mesenchymal translation in A431 and SCL-1 cells. NPTX2 interacted with METTL3, increased METTL3 expression, and improved N6-methyladenosine modification in cSCC cell lines. Mechanistically, NPTX2 facilitated epithelial‒mesenchymal translation by promoting METTL3-mediated N6-methyladenosine of SNAIL. METTL3 knockdown and N6-methyladenosine inhibition reversed the impacts of NPTX2 overexpression on cSCC cells. Invivo studies verified the role of NPTX2 as an oncogene in cSCC. Therefore, NPTX2 may be a potential therapeutic target for cSCC.

TEAD1 Silencing Regulates Cell Proliferation and Resistance to 5-Fluorouracil in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a skin malignant tumor account for approximately one-third of all nonmelanoma skin cancers. Studies have shown that TEA domain transcription factor 1 (TEAD1) is discovered to be involved in the pathogenesis of some human cancers, but to our knowledge its role in cSCC has not been reported. Samples from 16 cSCC patients and 27 healthy individuals were obtained for immunohistochemical staining of TEAD1. The expressions of TEAD1 in SCL-1, HSC-1 cells compared with the primary neonatal human epithelial keratinocytes were detected by Western blot and RT-qPCR. Proliferation and cell cycle of TEAD1 knockdown in cSCC cell lines were examined by MTT and flow cytometry analysis. Annexin V/PI and JC-1 staining were used to determine the cell apoptosis. The expression of TEAD1 decreased significantly in cSCC compared to its expression in normal skin tissues and cell lines. Down-regulation of TEAD1 in cSCC cell lines promoted cell growth via regulation of the G2/M progression. Additionally, silence of TEAD1 also protected cells against 5-Fluorouracil-induced apoptosis and decreased the expression of apoptosis-related protein (p53). Our results suggested that TEAD1 expression is down-regulated and functioned as a tumor suppressor in cSCC and that it may serve as a biomarker or therapeutic target of cSCC.

465 Super enhancer regulated LINC00094 upregulates the expression of metalloproteinases MMP-1 and MMP-13 and promotes invasion of cutaneous squamous cell carcinoma

Super enhancers have been shown to present a role in tumorigenesis, indicating that they could be promising therapeutic targets for cancer treatment. Long noncoding RNAs (lncRNAs) have emerged as important regulators of cancer progression. Here, the role of lncRNA LINC00094 in cutaneous squamous carcinoma (cSCC) was examined. Elevated LINC00094 expression was detected in cSCC cell lines, compared with normal human epidermal keratinocytes. The expression of LINC00094 in cSCC cells was downregulated by super enhancer inhibitors THZ1 and JQ1.

441 MiR-23b functions as a tumor suppressor in cutaneous squamous cell carcinoma and targets Ras-related 2 (RRAS2)

Cutaneous squamous cell carcinoma (cSCC) is one of the most common cancer types with metastatic potential. MicroRNAs (miRNAs) are small RNAs that regulate gene expression at the posttranscriptional level. Here, we report that miR-23b, an evolutionarily conserved miRNA that is abundantly expressed in keratinocytes, is downregulated in cSCCs and to a lesser extent in precancerous lesions. We show that miR-23b expression is regulated by the MAPK-signaling pathway. CRISPR/Cas9-mediated deletion of the MIR23B gene in cSCC cell lines resulted in increased colony formation and tumor sphere formation ability.