Growth and Viability of Cutaneous Squamous Cell Carcinoma Cell Lines Display Different Sensitivities to Isoform-Specific Phosphoinositide 3-Kinase Inhibitors

Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors.     

Increased Expression of Flightless I in Cutaneous Squamous Cell Carcinoma Affects Wnt/β-Catenin Signaling Pathway

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/β-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/β-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased β-catenin and a decrease in the expression of the downstream effector of β-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.     

Impaired Wound Healing,Fibrosis, and Cancer: The Paradigm of Recessive Dystrophic Epidermolysis Bullosa

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a devastating skin blistering disease caused by mutations in the gene encoding type VII collagen (C7), leading to epidermal fragility, trauma-induced blistering, and long term, hard-to-heal wounds. Fibrosis develops rapidly in RDEB skin and contributes to both chronic wounds, which emerge after cycles of repetitive wound and scar formation, and squamous cell carcinoma—the single biggest cause of death in this patient group. The molecular pathways disrupted in a broad spectrum of fibrotic disease are also disrupted in RDEB, and squamous cell carcinomas arising in RDEB are thus far molecularly indistinct from other sub-types of aggressive squamous cell carcinoma (SCC). Collectively these data demonstrate RDEB is a model for understanding the molecular basis of both fibrosis and rapidly developing aggressive cancer. A number of studies have shown that RDEB pathogenesis is driven by a radical change in extracellular matrix (ECM) composition and increased transforming growth factor-beta (TGFβ) signaling that is a direct result of C7 loss-of-function in dermal fibroblasts. However, the exact mechanism of how C7 loss results in extensive fibrosis is unclear, particularly how TGFβ signaling is activated and then sustained through complex networks of cell-cell interaction not limited to the traditional fibrotic protagonist, the dermal fibroblast. Continued study of this rare disease will likely yield paradigms relevant to more common pathologies.     

Molecular Mechanisms of Cutaneous Squamous Cell Carcinoma

Non-melanoma skin cancers are cutaneous malignancies representing the most common form of cancer in the United States. They are comprised predominantly of basal cell carcinomas and squamous cell carcinomas (cSCC). The incidence of cSCC is increasing, resulting in substantial morbidity and ever higher treatment costs; currently in excess of one billion dollars, per annum. Here, we review research defining the molecular basis and development of cSCC that aims to provide new insights into pathogenesis and drive the development of novel, cost and morbidity saving therapies.     

Comprehensive Mutational and Phenotypic Characterization of New Metastatic Cutaneous Squamous Cell Carcinoma Cell Lines Reveal Novel Drug Susceptibilities

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who develop metastases (2–5%) present with advanced disease that requires a combination of radical surgery and adjuvant radiation therapy. There are few effective therapies for refractory disease. In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically resembled the original patient tumor and were tumorogenic in mice. Differences in cancer-related gene expression between the tumor and cell lines after various culturing conditions could be largely reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries, which correlate with either copy number variations, targetable mutations and/or the upregulation of gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines are thus useful preclinical models for determining targetable pathways and candidate therapeutics.     

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.     

Immune Checkpoint Blockade in Advanced Cutaneous Squamous Cell Carcinoma: What Do We Currently Know in 2020?

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer that predominantly arises in chronically sun-damaged skin. Immunosuppression, genetic disorders such as xeroderma pigmentosum (XP), exposure to certain drugs and environmental noxae have been identified as major risk factors. Surgical removal of cSCC is the therapy of choice and mostly curative in early stages. However, a minority of patients develop locally advanced tumors or distant metastases that are still challenging to treat. Immune checkpoint blockade (ICB) targeting CTLA-4, PD-L1 and PD-1 has tremendously changed the field of oncological therapy and especially the treatment of skin cancers as tumors with a high mutational burden. In this review, we focus on the differences between cSCC and cutaneous melanoma (CM) and their implications on therapy, summarize the current evidence on ICB for the treatment of advanced cSCC and discuss the chances and pitfalls of this therapy option for this cancer entity. Furthermore, we focus on special subgroups of interest such as organ transplant recipients, patients with hematologic malignancies, XP and field cancerization.     

hTERT-Driven Immortalization of RDEB Fibroblast and Keratinocyte Cell Lines Followed by Cre-Mediated Transgene Elimination

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased β-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.     

High ROS Production by Celecoxib and Enhanced Sensitivity for Death Ligand-Induced Apoptosis in Cutaneous SCC Cell Lines

Incidence of cutaneous squamous cell carcinoma (cSCC) and actinic keratosis has increased worldwide, and non-steroidal anti-inflammatory drugs as celecoxib are considered for treatment. We show here strong anti-proliferative effects of celecoxib in four cSCC cell lines, while apoptosis and cell viability largely remained unaffected. Impeded apoptosis was overcome in combinations with agonistic CD95 antibody or TNF-related apoptosis-inducing ligand (TRAIL), resulting in up to 60% apoptosis and almost complete loss of cell viability. Proapoptotic caspase cascades were activated, and apoptosis was suppressed by caspase inhibition. TRAIL receptor (DR5) and proapoptotic Bcl-2 proteins (Puma and Bad) were upregulated, while anti-apoptotic factors (survivin, XIAP, cFLIP, Mcl-1, and Bcl-w) were downregulated. Strongly elevated levels of reactive oxygen species (ROS) turned out as particularly characteristic for celecoxib, appearing already after 2 h. ROS production alone was not sufficient for apoptosis induction but may play a critical role in sensitizing cancer cells for apoptosis and therapy. Thus, the full therapeutic potential of celecoxib may be better used in combinations with death ligands. Furthermore, the immune response against cSCC/AK may be improved by celecoxib, and combinations with checkpoint inhibitors, recently approved for the treatment of cSCC, may be considered.     

Less Expression of Prohibitin Is Associated with Increased Paired Box 2 (PAX2) in Renal Interstitial Fibrosis Rats

Prohibitin (PHB) and paired box 2 (PAX2) are associated with the development of renal interstitial fibrosis (RIF). This study was performed to investigate whether or not the PHB could regulate the PAX2 gene expression in unilateral ureteral obstruction (UUO) in rats. Eighty Wistar male rats were randomly divided into two groups: sham operation group (SHO) and model group subjected to unilateral ureteral obstruction (GU), n = 40, respectively. The model was established by left ureteral ligation. Renal tissues were collected at 14-day and 28-day after surgery. RIF index, protein expression of PHB, PAX2, transforming growth factor-βl (TGF-β1), α-smooth muscle actin (α-SMA), collagen-IV (Col-IV), fibronectin (FN) or cleaved Caspase-3, and cell apoptosis index in renal interstitium, and mRNA expressions of PHB, PAX2 and TGF-β1 in renal tissue were detected. When compared with those in SHO group, expression of PHB (mRNA and protein) was significantly reduced, and expressions of PAX2 and TGF-β1 (protein and mRNA) were markedly increased in the GU group (each p < 0.01). Protein expressions of α-SMA, Col-IV, FN and cleaved Caspase-3, and RIF index or cell apoptosis index in the GU group were markedly increased when compared with those in the SHO group (each p < 0.01). The protein expression of PHB was negatively correlated with protein expression of PAX2, TGF-β1, α-SMA, Col-IV, FN or cleaved Caspase-3, and RIF index or cell apoptosis index (all p < 0.01). In conclusion, less expression of PHB is associated with increased PAX2 gene expression and RIF index in UUO rats, suggesting that increasing the PHB expression is a potential therapeutic target for prevention of RIF.     

Foam Cells as Therapeutic Targets in Atherosclerosis with a Focus on the Regulatory Roles of Non-Coding RNAs

Atherosclerosis is a major cause of human cardiovascular disease, which is the leading cause of mortality around the world. Various physiological and pathological processes are involved, including chronic inflammation, dysregulation of lipid metabolism, development of an environment characterized by oxidative stress and improper immune responses. Accordingly, the expansion of novel targets for the treatment of atherosclerosis is necessary. In this study, we focus on the role of foam cells in the development of atherosclerosis. The specific therapeutic goals associated with each stage in the formation of foam cells and the development of atherosclerosis will be considered. Processing and metabolism of cholesterol in the macrophage is one of the main steps in foam cell formation. Cholesterol processing involves lipid uptake, cholesterol esterification and cholesterol efflux, which ultimately leads to cholesterol equilibrium in the macrophage. Recently, many preclinical studies have appeared concerning the role of non-encoding RNAs in the formation of atherosclerotic lesions. Non-encoding RNAs, especially microRNAs, are considered regulators of lipid metabolism by affecting the expression of genes involved in the uptake (e.g., CD36 and LOX1) esterification (ACAT1) and efflux (ABCA1, ABCG1) of cholesterol. They are also able to regulate inflammatory pathways, produce cytokines and mediate foam cell apoptosis. We have reviewed important preclinical evidence of their therapeutic targeting in atherosclerosis, with a special focus on foam cell formation.     

Evaluating a Targeted Cancer Therapy Approach Mediated by RNA trans-Splicing In Vitro and in a Xenograft Model for Epidermolysis Bullosa-Associated Skin Cancer

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.     

A Gene Expression Signature to Predict Nucleotide Excision Repair Defects and Novel Therapeutic Approaches

Nucleotide excision repair (NER) resolves DNA adducts, such as those caused by ultraviolet light. Deficient NER (dNER) results in a higher mutation rate that can predispose to cancer development and premature ageing phenotypes. Here, we used isogenic dNER model cell lines to establish a gene expression signature that can accurately predict functional NER capacity in both cell lines and patient samples. Critically, none of the identified NER deficient cell lines harbored mutations in any NER genes, suggesting that the prevalence of NER defects may currently be underestimated. Identification of compounds that induce the dNER gene expression signature led to the discovery that NER can be functionally impaired by GSK3 inhibition, leading to synergy when combined with cisplatin treatment. Furthermore, we predicted and validated multiple novel drugs that are synthetically lethal with NER defects using the dNER gene signature as a drug discovery platform. Taken together, our work provides a dynamic predictor of NER function that may be applied for therapeutic stratification as well as development of novel biological insights in human tumors.     

Expression Profiles of ASIC1/2 and TRPV1/4 in Common Skin Tumors

The acid-sensing ion channels ASIC1 and ASIC2, as well as the transient receptor potential vanilloid channels TRPV1 and TRPV4, are proton-gated cation channels that can be activated by low extracellular pH (pH_e), which is a hallmark of the tumor microenvironment in solid tumors. However, the role of these channels in the development of skin tumors is still unclear. In this study, we investigated the expression profiles of ASIC1, ASIC2, TRPV1 and TRPV4 in malignant melanoma (MM), squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and in nevus cell nevi (NCN). We conducted immunohistochemistry using paraffin-embedded tissue samples from patients and found that most skin tumors express ASIC1/2 and TRPV1/4. Striking results were that BCCs are often negative for ASIC2, while nearly all SCCs express this marker. Epidermal MM sometimes seem to lack ASIC1 in contrast to NCN. Dermal portions of MM show strong expression of TRPV1 more frequently than dermal NCN portions. Some NCN show a decreasing ASIC1/2 expression in deeper dermal tumor tissue, while MM seem to not lose ASIC1/2 in deeper dermal portions. ASIC1, ASIC2, TRPV1 and TRPV4 in skin tumors might be involved in tumor progression, thus being potential diagnostic and therapeutic targets.     

Isolation and Characterization of Squamous Cell Carcinoma-Derived Stem-like Cells: Role in Tumor Formation

In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of β₁-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2–4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.     

Suppression of Lysophosphatidylcholine-Induced Human Aortic Smooth Muscle Cell Calcification by Protein Kinase A Inhibition

Lysophosphatidylcholine (lysoPtdCho) is produced mainly by the phospholipase A2-dependent hydrolysis of phosphatidylcholine (PtdCho) and can induce inflammatory activation and osteogenic gene expression in vascular smooth muscle cells. However, the mechanisms mediating these processes have not been fully elucidated. In this study, we investigated whether inhibition of protein kinase A (PKA) signaling suppressed lysoPtdCho-induced calcification of human aortic smooth muscle cells (HASMC). Calcium levels and alkaline phosphatase activity were significantly increased in HASMC treated with lysoPtdCho, but not PtdCho, compared with those in phosphate-buffered saline-treated HASMC. However, the addition of a PKA inhibitor (H-89) or PKA siRNA blocked lysoPtdCho-induced HASMC calcification. These results showed that lysoPtdCho could activate PKA-mediated HASMC calcification and that PKA may be a therapeutic target for lysoPtdCho-mediated vascular smooth muscle cell calcification.     

A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System

Tyrosine kinase substrate with four SH3 domains (Tks4) scaffold protein plays roles in cell migration and podosome formation and regulates systemic mechanisms such as adult bone homeostasis and adipogenesis. Mutations in the Tks4 gene (SH3PXD2b) cause a rare developmental disorder called Frank-Ter Haar syndrome (FTHS), which leads to heart abnormalities, bone tissue defects, and reduced adiposity. We aimed to produce a human stem cell-based in vitro FTHS model system to study the effects of the loss of the Tks4 protein in different cell lineages and the accompanying effects on the cell signalome. To this end, we used CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas9)) to knock out the SH3PXD2b gene in the HUES9 human embryonic stem cell line (hESC), and we obtained stable homo- and heterozygous knock out clones for use in studying the potential regulatory roles of Tks4 protein in embryonic stem cell biology. Based on pluripotency marker measurements and spontaneous differentiation capacity assays, we concluded that the newly generated Tks4-KO HUES9 cells retained their embryonic stem cell characteristics. We propose that the Tks4-KO HUES9 cells could serve as a tool for further cell differentiation studies to investigate the involvement of Tks4 in the complex disorder FTHS. Moreover, we successfully differentiated all of the clones into mesenchymal stem cells (MSCs). The derived MSC cultures showed mesenchymal morphology and expressed MSC markers, although the expression levels of mesodermal and osteogenic marker genes were reduced, and several EMT (epithelial mesenchymal transition)-related features were altered in the Tks4-KO MSCs. Our results suggest that the loss of Tks4 leads to FTHS by altering cell lineage differentiation and cell maturation processes, rather than by regulating embryonic stem cell potential.     

In Vitro and In Vivo Effects of the Urokinase Plasminogen Activator Inhibitor WX-340 on Anaplastic Thyroid Cancer Cell Lines

Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.