The Molecular Mechanisms Of SHARPIN’s Functions On The Development Of Cutaneous Squamous Cell Carcinoma

BackgroundSHARPIN(also known as SIPL1)was originally identified as a SHANK(SH3(Src homology 3)and multiple ankyrin repeat domains protein)-binding protein enriched in the postsynaptic density of excitatory neurotransmitters.SHARPIN has been detected in brain,spleen,lungs,and certain cancer types.Earlier studies identified Sharpin as the gene mutated in the chronic proliferative dermatitis mice(cpdm),which spontaneously develop severe chronic inflammation primarily in the skin but also in other tissues such as the gut,lung,liver,and esophagus.SHARPIN was recently identified as a common part of the linear ubiquitin chain assembly complex(LUBAC)which also contains RNF31(HOIP)and RBCK1(HOIL-1L).LUBAC is an important component of the NF-κB and JNK signaling pathway which is a critical regulator of inflammation,the immune response,and lymphoid tissue development.SHARPIN has been identified as a commonly overexpressed pro-oncogene and functionally plays tumor-associated roles during cancer progression in recent studies.SHARPIN promotes the migration of CHO cells in vitro and lymphocytes in vivo,and enhances the lung metastasis of osteosarcoma in vivo(in immunocompromised mice).Upregulation of SHARPIN was observed in ovarian cancer,renal cell carcinoma,cervical cancer and prostate cancer.Furthermore,SHARPIN was reported to inhibit PTEN via a physical interaction.SHARPIN could inactivate integrins in many cell types and influence integrin dependent cellular functions.SHARPIN was identified as a metastasis gene in breast cancer(BC)by using a replication-incompetent gammaretroviral vector(yRV),which could be a potential biomarker for stratifying BC patients.Collectively,evidence demonstrates a role of SHARPIN in promoting tumorigenesis.Cutaneous Squamous Cell Carcinoma(cSCC)is a common skin cancer characterized by the malignant proliferation of keratinizing cells of the epidermis or its appendages.cSCC usually arises from precursor lesions such as actinic keratosis,and Bowen’s disease(SCC in situ).cSCC is the second most common non-melanoma skin cancers(NMSCs)globally,with an increasing incidence,which accounts for one million new cases diagnosed annually in the US.The rate of metastasis in cSCC has been estimated to range from 2%to 5%.The three major groups of environmental or host-dependent risk factors identified for cSCC are ultraviolet radiation(UVR)exposure,genetic predisposition and immunosuppression.The transformation of a normal keratinocyte into a promalignant AK,BD,invasive cSCC and metastasis is likely to be a multiple process requiring the accumulation of somatic genetic and epigenetic alternations in key signaling pathways regulating cell fate,cell survival and genome maintenance.The advent of massively parallel(so called ’next generation’)sequencing techniques is both facilitating and complicating this field by demonstrating a very high burden of mutations in cSCC,which makes determination of driver genes daunting,particularly in terms of distinguishing them from passenger mutations that may have minimal impact on tumor progression.Published research has particularly focused on TP53,NOTCH,RAS,EGFR,SRC-family kinase(SFK),CDKN2A,NF-KB,TGFβ and,most recently,KNSTRN.Evidence has been presented that alterations in the microenvironment of the premalignant lesion,including the composition of the epidermal basement membrane and dermal extracellular matrix(ECM),the influx of inflammatory cells,and the presence of microbial structures,promote the growth and progression of cSCC.Matrix metalloproteinases(MMPs)released by tumor cells,stromal cancer-associated fibroblasts,and inflammatory cells support tumor growth and invasion by remodeling the basement membrane and stromal ECM.MMPs also regulate the activity and availability of growth factors,chemokines,and cytokines in the tumor microenvironment and may,in this way,promote inflammation and cancer progression.pDCs(plasmacytoid dendritic cells)have been shown to be a component of the perineoplastic inflammatory infiltrate in cSCC.pDCs have the potential to contribute to anti-tumor immunity;however,accumulating evidence suggests that the complex interaction of pDCs with tumor cells and their micro-environment appears to contribute to immunologic tolerance.The current understanding of the role of pDCs against cutaneous malignancies has therapeutic implications.LCs(Langerins cells,LCs)are involved in the metabolism of DMBA to its mutagenic forms and LC-deficient huLangerin-diphtheria toxin A(Lang-DTA)mice showed almost complete resistance to DMBA-TPA-induced cutaneous carcinogenesis.TNF-α and IL-1 can induce auto-inflammation,leading to jury,stimulating cell proliferation,and promoting angiogenesis and tumor invasion.Treatment options currently available for locally advanced or metastatic cSCCs have limited effects on overall survival and new systemic treatments are badly needed.Collectively,these backgrounds promoted us to investigate the expression and mutations of SHARPIN in skin tumors and identify a promising biomarker and therapeutic target for NMSCs.Objectives1.To measure the mutations of SHARPIN in cSCC tissues;2.To evaluate the expression of SHARPIN in cSCC tissues;3.To explore the role of SHARPIN in regulating proliferation,apoptosis,and cell cycle of A431;4.To investigate the potential molecules and pathways underlying the regulation of SHARPIN on cSCC.Methods1.The expression of SHARPIN in cSCC1)The expression of SHARPIN in cSCC tissues was measured by immunohistochemistry;2)The location of SHARPIN in cSCC tissues was detected by immunofluorescence.2.The mutation of SHARPIN in cSCCThe DNA was extracted from formalin-fixed paraffin embedded blocks and the 8 exons and flanking sequences of SHARPIN were amplified by PCR,which was followed by Sanger sequencing.3.The regulation of SHARPIN on the functions of A4311)The lentivirus vectors to overexpress and inhibit SHARPIN were constructed and transfected into A431.The efficiency of transfection was evaluated by Western Blot and qRT-PCR;2)The effects of SHARPIN on proliferation of A431 was determined by EdU;3)The effects of SHARPIN on apoptosis of A431 was determined by TUNEL;4)The effects of SHARPIN on cell cycle of A431 was determined by flowcytometry;4.The potential pathways underlying the regulation of SHARPIN on cSCC1)After treated with TNF-α,key molecules in NF-κB,JNK,and NOTCH1 signaling pathways were detected by Western Blot;2)The spectrum of gene expression changes in overexpression and inhibition groups of A431 was characterized by transcriptome sequencing,which may provide an insight into the potential molecular mechanisms.Results1.In some cSCC tissues,SHARPIN was absent in the cancer nest and in the peri-tumor areas,its expression was significantly decreased compared with the high expression of SHARPIN in normal skin tissues and keratoananthoma.From the cancer nests,peri-tumor areas of cSCC to normal skins,the expression of SHARPIN decreased gradually.In general,68.1%of cSCC had low expression of SHARPIN and 31.9%had mediate expression.In 36.2%of cSCC,the expression of SHARPIN was nearly absolutely lost.The results of immunofluorescence suggested that SHARPIN was mainly located in the epidermis and in the cytoplasma.2.Total 47 cSCC cases were collected and the results of sequencing indicated that the mutation rate of SHARPIN was 17.0%and the C>T transition rate was 6.4%.3.The proliferation rate,apoptosis rate and the proportion of cells in each stages of cell cycle did not show significant difference in statistics.4.Key molecules in NF-κB,JNK,and NOTCH1 signaling pathways did show significant changes.5.The results of transcriptome sequencing suggested that the most significantly changed gene was ITGA4 which had the closest relationship with SHARPIN.Conclusions1.The expression of SHARPIN was significantly decreased and even lost in cSCC,which had a gradually decreasing trend from normal skin to peri-tumor areas and cancer nests.There was relative high rate of mutations of SHARPIN in cSCC,which indicated that SHARPIN might act as a tumor suppressor.2.SHARPIN was not involved in the regulation of proliferation,apoptosis,and cell cycle of A431.3.SHARPIN had no effects on NF-κB,JNK,and NOTCH1 signaling pathways in A431.4.There might be interaction between SHARPIN and ITGA4,which might regulate the angiogenesis,invasion and metastasis of A431.