The Identification of Potential Therapeutic Targets for Cutaneous Squamous Cell Carcinoma.

We performed a small interfering RNA screen to identify targets for cutaneous squamous cell carcinoma (cSCC) therapy in the ubiquitin/ubiquitin-like system. We provide evidence for selective anti-cSCC activity of knockdown of the E3 ubiquitin ligase MARCH4, the ATPase p97/VCP, the deubiquitinating enzyme USP8, the cullin-RING ligase (CRL) 4 substrate receptor CDT2/DTL, and components of the anaphase-promoting complex/cyclosome (APC/C). Specifically attenuating CRL4CDT2 by CDT2 knockdown can be more potent in killing cSCC cells than targeting CRLs or CRL4s in general by RBX1 or DDB1 depletion. Suppression of the APC/C or forced APC/C activation by targeting its repressor EMI1 are both potential therapeutic approaches. We observed that cSCC cells can be selectively killed by small-molecule inhibitors of USP8 (DUBs-IN-3/compound 22c) and the NEDD8 E1 activating enzyme/CRLs (MLN4924/pevonedistat). A substantial proportion of cSCC cell lines are very highly MLN4924-sensitive. Pathways that respond to defects in proteostasis are involved in the anti-cSCC activity of p97 suppression. Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development. EMI1 and CDT2 depletion can selectively cause DNA re-replication and DNA damage in cSCC cells.

Targeting the spliceosome for cutaneous squamous cell carcinoma therapy: a role for c-MYC and wild-type p53 in determining the degree of tumour selectivity.

We show that suppression of the spliceosome has potential for the treatment of cutaneous squamous cell carcinoma (cSCC). The small-molecule inhibitors of the spliceosome at the most advanced stage of development target the splicing factor SF3B1/SF3b155. The majority of cSCC cell lines are more sensitive than normal skin cells to death induced by the SF3B1 inhibitor pladienolide B. Knockdown of SF3B1 and a range of other splicing factors with diverse roles in the spliceosome can also selectively kill cSCC cells. We demonstrate that endogenous c-MYC participates in conferring sensitivity to spliceosome inhibition. c-MYC expression is elevated in cSCC lines and its knockdown reduces alterations in mRNA splicing and attenuates cell death caused by interference with the spliceosome. In addition, this study provides further support for a key role of the p53 pathway in the response to spliceosome disruption. SF3B1 inhibition causes wild-type p53 upregulation associated with altered mRNA splicing and reduced protein expression of both principal p53 negative regulators MDMX/MDM4 and MDM2. We observed that wild-type p53 can promote pladienolide B-induced death in tumour cells. However, p53 is commonly inactivated by mutation in cSCCs and p53 participates in killing normal skin cells at high concentrations of pladienolide B. This may limit the therapeutic window of SF3B1 inhibitors for cSCC. We provide evidence that, while suppression of SF3B1 has promise for treating cSCCs with mutant p53, inhibitors which target the spliceosome through SF3B1-independent mechanisms could have greater cSCC selectivity as a consequence of reduced p53 upregulation in normal cells.

Preclinical comparison of proteasome and ubiquitin E1 enzyme inhibitors in cutaneous squamous cell carcinoma: the identification of mechanisms of differential sensitivity.

Proteasome inhibitors have distinct properties and the biochemical consequences of suppressing ubiquitin E1 enzymes and the proteasome differ. We compared the effects of the proteasome inhibitors bortezomib, ixazomib and carfilzomib and the ubiquitin E1 enzyme inhibitor MLN7243/TAK-243 on cell viability and cell death in normal keratinocytes and cutaneous squamous cell carcinoma (cSCC) cell lines. The effects of both a pulse of treatment and more extended incubation were investigated. This is relevant to directly-delivered therapy (topical treatment/intratumoral injection) where the time of exposure can be controlled and a short exposure may better reflect systemically-delivered inhibitor pharmacokinetics. These agents can selectively kill cSCC cells but there are variations in the pattern of cSCC cell line sensitivity/resistance. Variations in the responses to proteasome inhibitors are associated with differences in the specificity of the inhibitors for the three proteolytic activities of the proteasome. There is greater selectivity for killing cSCC cells compared to normal keratinocytes with a pulse of proteasome inhibitor treatment than with a more extended exposure. We provide evidence that c-MYC-dependent NOXA upregulation confers susceptibility to a short incubation with proteasome inhibitors by priming cSCC cells for rapid BAK-dependent death. We observed that bortezomib-resistant cSCC cells can be sensitive to MLN7243-induced death. Low expression of the ubiquitin E1 UBA1/UBE1 participates in conferring susceptibility to MLN7243 by increasing sensitivity to MLN7243-mediated attenuation of ubiquitination. This study supports further investigation of the potential of proteasome and ubiquitin E1 inhibition for cSCC therapy. Direct delivery of inhibitors could facilitate adequate exposure of skin cancers.

Inactivation of TGFß receptors in stem cells drives cutaneous squamous cell carcinoma.

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFß Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through Braf(V600E) or Kras(G12D) knockin) and TGFß signalling ablation (through Tgfbr1 deletion) in LGR5(+ve) stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5(+ve) cells also results in cSCC development. These findings indicate that LGR5(+ve) stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFß signalling, are driving events of skin tumorigenesis.

Novel CARD11 Mutations in Human Cutaneous Squamous Cell Carcinoma Lead to Aberrant NF-κB Regulation.

NF-κB signaling plays a crucial role in regulating proliferation and differentiation in the epidermis. Alterations in the NF-κB pathway can lead to skin pathologies with a significant burden to human health such as psoriasis and cutaneous squamous cell carcinoma (cSCC). Caspase recruitment domain (CARD)-containing scaffold proteins are key regulators of NF-κB signaling by providing a link between membrane receptors and NF-κB transcriptional subunits. Mutations in the CARD family member, CARD14, have been identified in patients with the inflammatory skin diseases psoriasis and pityriasis rubra pilaris. Here, we describe that the gene coding for another CARD scaffold protein, CARD11, is mutated in more than 38% of 111 cSCCs, and show that novel variants outside of the coiled-coil domain lead to constitutively activated NF-κB signaling. CARD11 protein expression was detectable in normal skin and increased in all cSCCs tested. CARD11 mRNA levels were comparable with CARD14 in normal skin and CARD11 mRNA was increased in cSCC. In addition, we identified CARD11 mutations in peritumoral and sun-exposed skin, suggesting that CARD11-mediated alterations in NF-κB signaling may be an early event in the development of cSCC.

NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.

Cutaneous SCC (cSCC) is the most frequently occuring skin cancer with metastatic potential and can manifest rapidly as a common side effect in patients receiving systemic kinase inhibitors. Here, we use massively parallel exome and targeted level sequencing of 132 sporadic cSCCs and of 39 squamoproliferative lesions and cSCCs arising in patients receiving the BRAF inhibitor vemurafenib, as well as 10 normal skin samples, to identify NOTCH1 mutation as an early event in squamous cell carcinogenesis. Bisected vemurafenib-induced lesions revealed surprising heterogeneity with different activating HRAS and NOTCH1 mutations identified in two halves of the same cSCC, suggesting polyclonal origin. Immunohistochemical analysis using an antibody specific to nuclear NOTCH1 correlates with mutation status in sporadic cSCCs, and regions of NOTCH1 loss or downregulation are frequently observed in normal-looking skin. Our data indicate that NOTCH1 acts as a gatekeeper in human cSCC.

Methylated tissue factor pathway inhibitor 2 (TFPI2) DNA in serum is a biomarker of metastatic melanoma.

Transcriptional silencing of tissue factor pathway inhibitor 2 (TFPI2) occurs in several human tumors including melanoma. We investigated methylated TFPI2 as a biomarker of metastatic melanoma using qRT-PCR to assess TFPI2 expression and pyrosequencing to analyze CpG island methylation in malignant melanoma cell lines, in benign nevi, in 112 primary and metastatic melanomas, and in serum from 6 healthy individuals and 35 patients: 20 patients with primary and 15 patients with metastatic melanoma. The TFPI2 CpG island is unmethylated in nevi but methylation is associated with metastatic melanoma. Circulating methylated TFPI2 DNA is undetectable in sera from healthy individuals and detectable in sera from patients with primary and metastatic melanomas, but the presence of methylated TFPI2 DNA in serum is strongly associated with metastatic disease (P<0.01). Detection of TFPI2-methylated DNA in the serum of patients with resected melanoma is a sensitive and specific biomarker of metastatic melanoma. Confirmation of our results in independent patient cohorts would encourage prospective evaluation as a biomarker of disease state.

Nodular granulomatous phlebitis: a phlebitic tuberculid.

A 22-year-old woman presented with recurrent non-ulcerating skin nodules overlying the great saphenous vein on the anteromedial lower legs. Histology showed a granulomatous phlebitis, and polymerase chain reaction performed on lesional skin detected DNA specific for Mycobacterium tuberculosis. The lesions resolved with anti-tuberculous therapy. This case may be a further example of nodular granulomatous phlebitis, a phlebitic tuberculid.

Real-time GUS analysis using Q-PCR instrumentation.

The development of new technology within biological sciences has resulted in a number of real-time PCR instruments that have become essential tools within molecular biology. This equipment has facilitated high throughput analysis of samples and optimal information gathering of completed PCR reactions for example estimating the copy number of a gene of interest that is inserted into particular genomes. Real-time PCR instruments frequently come with optional filter sets, e.g. the ALEXA filter set which has parameters in common with excitation and emission wavelengths of sodium methyl umbelliferone (NaMU) widely used in beta-glucuronidase reporter gene assays. Using these filter sets it has been possible to quantify and measure gus A activity of Ulmus procera SR4 in real-time removing the necessity for aliquots of reactions to be stopped by pipetting into carbonate buffer for each time point. The introduction of real-time GUS analysis leads to faster, more accurate and reproducible assays with reduced potential for pipetting errors, requires fewer manipulations and encourages high throughput analysis of inter-individual gene expression variation.