The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome.

Epigenetic mechanisms oversee epidermal homeostasis and oncogenesis. The identification of kinases controlling these processes has direct therapeutic implications. We show that ULK3 is a nuclear kinase with elevated expression levels in squamous cell carcinomas (SCCs) arising in multiple body sites, including skin and Head/Neck. ULK3 loss by gene silencing or deletion reduces proliferation and clonogenicity of human keratinocytes and SCC-derived cells and affects transcription impinging on stem cell-related and metabolism programs. Mechanistically, ULK3 directly binds and regulates the activity of two histone arginine methyltransferases, PRMT1 and PRMT5 (PRMT1/5), with ULK3 loss compromising PRMT1/5 chromatin association to specific genes and overall methylation of histone H4, a shared target of these enzymes. These findings are of translational significance, as downmodulating ULK3 by RNA interference or locked antisense nucleic acids (LNAs) blunts the proliferation and tumorigenic potential of SCC cells and promotes differentiation in two orthotopic models of skin cancer.

Cutaneous Surgical Wounds Have Distinct Microbiomes from Intact Skin.

Infections are relatively rare following cutaneous surgical procedures, despite the potential for wound exposure to pathogens both during surgery and throughout the healing process. Although gut commensals are believed to reduce the risk of intestinal infections, an analogous role for skin commensals has not been described. In fact, the microbiome of normally healing surgical skin wounds has not yet been profiled using culture-independent techniques. We characterized the wound microbiome in 53 patients who underwent skin cancer surgery and healed without signs or symptoms of infection. A week after surgery, several bacterial species displayed significant differences in relative abundance when compared to control, nonoperated skin from the same patient. The relative abundance of the most common bacterium found on intact skin, Cutibacterium acnes, was reduced in wounds 5-fold. Staphylococcus aureus, a frequent cause of postoperative skin infections, was enriched 6.4-fold in clinically noninfected wounds, suggesting active suppression of pathogenicity. Finally, members of the Corynebacterium genus were the dominant organism in postoperative wounds, making up 37% of the average wound microbiome. The enrichment of these bacteria in normally healing wounds suggests that they might be capable of providing colonization resistance. Future studies focused on the biological and clinical significance of the wound microbiome may shed light on normal wound healing and potential therapeutic opportunities to mitigate infection risk. IMPORTANCE Commensal bacteria on skin may limit the ability of pathogenic bacteria to cause clinically significant infections. The bacteria on healing acute wounds, which might provide such a protective effect, have not been described using culture-independent approaches in the absence of antibiotics. We compare the microbiome of wounds a week after skin cancer removal surgery with intact skin from the same patient. We find that the potentially pathogenic species S. aureus is common on these healing wounds despite the absence of symptoms or signs of infection. We report that bacteria often considered as potential skin probiotics, including Staphylococcus epidermidis, do not reach high relative abundance in wound microbiomes. In contrast, specific members of the Corynebacterium genus, rarely associated with infections, were significantly enriched in healing wounds compared to intact skin. Future work is needed to see if Corynebacterium species or derivatives thereof could be employed to lower the risk of wound infection.

Imaging technologies for presurgical margin assessment of basal cell carcinoma.

Basal cell carcinoma is the most common cancer worldwide, necessitating the development of techniques to decrease treatment costs through efficiency and efficacy. Mohs micrographic surgery, a specialized surgical technique involving staged resection of the tumor with complete histologic evaluation of the peripheral margins, is highly utilized. Reducing stages by even 5% to 10% would result in significant improvement in care and economic benefits. Noninvasive imaging could aid in both establishing the diagnosis of suspicious skin lesions and streamlining the surgical management of skin cancers by improving presurgical estimates of tumor sizes. Herein, we review the current state of imaging techniques in dermatology and their applications for diagnosis and tumor margin assessment of basal cell carcinoma prior to Mohs micrographic surgery.

Lethal Dermal Sarcoma in Immunosuppressed Patients.

Skin cancer is the leading malignancy in immunosuppressed patients, including organ transplant recipients (OTRs), which is increasing in incidence as OTRs live longer. We performed a single-center case series of 4 patients with scalp pleomorphic dermal sarcoma and a history of multiple keratinocyte carcinomas. Outcomes included incidence of dermal sarcoma, dermal sarcoma-related mortality, and histopathologic findings. Out of more than 200 patients followed over a 3-year period in Massachusetts General Hospital High Risk Skin Cancer Clinics, all skin cancer-related deaths (2/2) were due to metastatic dermal sarcoma. Three of 4 patients diagnosed with scalp dermal sarcoma were OTRs and had been on at least one immunosuppressive medication for a median of 9 years. For patients who died from dermal sarcoma, the median time between diagnosis and death was 6 months. Our findings suggest pleomorphic dermal sarcoma contributes to skin cancer-related morbidity and mortality in OTRs.

Brigham and Women's Hospital tumor classification system for basal cell carcinoma identifies patients with risk of metastasis and death.

BACKGROUND: Despite approximately 4400 locally advanced US cases annually, high-stage basal cell carcinoma (BCC) is ill-defined. OBJECTIVE: To develop a tumor (T) staging system for BCC that will predict metastasis/death and compare its performance with that of the American Joint Committee on Cancer 8th edition (AJCC8) T-staging system. METHODS: Brigham and Women's Hospital (BWH) T staging was developed from a previously published nested cohort of 488 primary BCCs. Tumors were staged via BWH and AJCC8 T-staging systems, and predictions of metastasis and/or death were compared. RESULTS: The BWH and AJCC8 T-staging systems both captured all metastases/deaths in high T stages (BWH, T2; AJCC8, T3/T4). BWH T2 included 54% fewer cases ≥2 cm than AJCC8 T3/T4. BWH had a higher specificity (0.92 vs 0.80; P < .001) and positive predictive value (24% vs 11%, P < .001) for identifying cases at risk for metastasis/death, and the C-statistic was superior for BWH (P < .001). The BWH T2 10-year cumulative incidence of metastasis/death was 37% (95% confidence interval, 21%-60%). LIMITATIONS: Two-center cohort. CONCLUSIONS: BWH and AJCC 8 BCC staging both capture all metastases and deaths in the upper stages. However, BWH staging does so in half the number of cases, thus minimizing inappropriate up-staging. The risk of metastasis or death in BWH T2 BCC is sufficient to warrant surveillance for recurrence and clinical trials of adjuvant therapy.

NOTCH1 gene amplification promotes expansion of Cancer Associated Fibroblast populations in human skin.

Cancer associated fibroblasts (CAFs) are a key component of the tumor microenvironment. Genomic alterations in these cells remain a point of contention. We report that CAFs from skin squamous cell carcinomas (SCCs) display chromosomal alterations, with heterogeneous NOTCH1 gene amplification and overexpression that also occur, to a lesser extent, in dermal fibroblasts of apparently unaffected skin. The fraction of the latter cells harboring NOTCH1 amplification is expanded by chronic UVA exposure, to which CAFs are resistant. The advantage conferred by NOTCH1 amplification and overexpression can be explained by NOTCH1 ability to block the DNA damage response (DDR) and ensuing growth arrest through suppression of ATM-FOXO3a association and downstream signaling cascade. In an orthotopic model of skin SCC, genetic or pharmacological inhibition of NOTCH1 activity suppresses cancer/stromal cells expansion. Here we show that NOTCH1 gene amplification and increased expression in CAFs are an attractive target for stroma-focused anti-cancer intervention.

Dual-Wavelength Optical Polarization Imaging for Detecting Skin Cancer Margins.

Treatment of keratinocyte carcinomas requires an assessment of the extent of tumor spread. Visual delineation of tumor margins is error-prone owing to the limited contrast between cancerous and normal skin. In this contribution, we introduce spectrally-encoded optical polarization imaging and evaluate its performance for preoperative demarcation of keratinocyte carcinomas. Subjects with basal or squamous cell carcinoma, scheduled for Mohs surgery, were enrolled. The surgeon outlined the clinical boundary of each lesion preoperatively. Optical images of the lesions were then acquired at 440 and 640 nm. Spectral encoding of the experimental images minimized the impact of background pigmentation and vascularization. The surgeon was blinded to the imaging results. Margin assessments by imaging and by the surgeon were recorded and compared with the intraoperative histopathology. In total, 53 lesions were imaged in vivo. Thirteen cases required more than one Mohs stage. In all these cases, images accurately visualized the tumor. For cases negative following the first Mohs stage, margin assessments correlated with histopathology in 39 out of 40 cases. Imaging demonstrated 100% sensitivity and 98% specificity. Spectrally-encoded optical polarization imaging may prove valuable for real-time noninvasive preoperative delineation of skin cancer.

Factors predictive of recurrence, metastasis, and death from primary basal cell carcinoma 2 cm or larger in diameter.

BACKGROUND: Basal cell carcinoma (BCC) recurrence and metastatic rates are known to be very low. The risk factors for these rare outcomes are subsequently not well studied. OBJECTIVE: To identify risk factors independently associated with local recurrence (LR) and metastasis and/or death (M/D) in large (≥2 cm) BCC. METHODS: BCCs histologically confirmed between 2000 and 2009 were retrospectively screened for tumor diameter at 2 academic centers. Medical records of all large BCCs and an equal number of randomly selected small BCCs were reviewed for LR and M/D. RESULTS: Included were 248 large BCC and 248 small BCC tumors. Large BCCs had a significantly higher risk of LR and M/D than small BCCs (LR: 8.9% vs 0.8%, P < .001; M/D: 6.5% vs. 0%, P < .001). Because the risks were so low in small BCCs, they were excluded from further analysis. On multivariable logistic regression, head/neck location (odds ratio [OR], 9.7; 95% confidence interval [CI], 3.0-31.3) and depth beyond fat (OR, 3.1; 95% CI, 1.0-9.6) were associated with LR in large BCCs. Risk of LR was lower with Mohs micrographic surgery (OR, 0.14; 95% CI, 0.04-0.5). Head/neck location (OR, 5.3; 95% CI, 1.2-23.2), tumor diameter ≥4 cm (OR, 11.9; 95% CI, 2.4-59.4), and depth beyond fat (OR, 28.6; 95% CI, 6.7-121) were significant predictors of M/D in large BCCs. LIMITATIONS: Retrospective cohort design. CONCLUSIONS: Large BCCs, particularly those with additional risk factors, have a high enough risk of recurrence and metastasis to warrant further investigation to optimize management.

Immunity to commensal papillomaviruses protects against skin cancer.

Immunosuppression increases the risk of cancers that are associated with viral infection1. In particular, the risk of squamous cell carcinoma of the skin-which has been associated with beta human papillomavirus (ß-HPV) infection-is increased by more than 100-fold in immunosuppressed patients2-4. Previous studies have not established a causative role for HPVs in driving the development of skin cancer. Here we show that T cell immunity against commensal papillomaviruses suppresses skin cancer in immunocompetent hosts, and the loss of this immunity-rather than the oncogenic effect of HPVs-causes the markedly increased risk of skin cancer in immunosuppressed patients. To investigate the effects of papillomavirus on carcinogen-driven skin cancer, we colonized several strains of immunocompetent mice with mouse papillomavirus type 1 (MmuPV1)5. Mice with natural immunity against MmuPV1 after colonization and acquired immunity through the transfer of T cells from immune mice or by MmuPV1 vaccination were protected against skin carcinogenesis induced by chemicals or by ultraviolet radiation in a manner dependent on CD8+ T cells. RNA and DNA in situ hybridization probes for 25 commensal ß-HPVs revealed a significant reduction in viral activity and load in human skin cancer compared with the adjacent healthy skin, suggesting a strong immune selection against virus-positive malignant cells. Consistently, E7 peptides from ß-HPVs activated CD8+ T cells from unaffected human skin. Our findings reveal a beneficial role for commensal viruses and establish a foundation for immune-based approaches that could block the development of skin cancer by boosting immunity against the commensal HPVs present in all of our skin.

CSL controls telomere maintenance and genome stability in human dermal fibroblasts.

Genomic instability is a hallmark of cancer. Whether it also occurs in Cancer Associated Fibroblasts (CAFs) remains to be carefully investigated. Loss of CSL/RBP-Jκ, the effector of canonical NOTCH signaling with intrinsic transcription repressive function, causes conversion of dermal fibroblasts into CAFs. Here, we find that CSL down-modulation triggers DNA damage, telomere loss and chromosome end fusions that also occur in skin Squamous Cell Carcinoma (SCC)-associated CAFs, in which CSL is decreased. Separately from its role in transcription, we show that CSL is part of a multiprotein telomere protective complex, binding directly and with high affinity to telomeric DNA as well as to UPF1 and Ku70/Ku80 proteins and being required for their telomere association. Taken together, the findings point to a central role of CSL in telomere homeostasis with important implications for genomic instability of cancer stromal cells and beyond.

Dualism of FGF and TGF-ß Signaling in Heterogeneous Cancer-Associated Fibroblast Activation with ETV1 as a Critical Determinant.

Heterogeneity of cancer-associated fibroblasts (CAFs) can result from activation of distinct signaling pathways. We show that in primary human dermal fibroblasts (HDFs), fibroblast growth factor (FGF) and transforming growth factor ß (TGF-ß) signaling oppositely modulate multiple CAF effector genes. Genetic abrogation or pharmacological inhibition of either pathway results in induction of genes responsive to the other, with the ETV1 transcription factor mediating the FGF effects. Duality of FGF/TGF-ß signaling and differential ETV1 expression occur in multiple CAF strains and fibroblasts of desmoplastic versus non-desmoplastic skin squamous cell carcinomas (SCCs). Functionally, HDFs with opposite TGF-ß versus FGF modulation converge on promoting cancer cell proliferation. However, HDFs with increased TGF-ß signaling enhance invasive properties and epithelial-mesenchymal transition (EMT) of SCC cells, whereas HDFs with increased FGF signaling promote macrophage infiltration. The findings point to a duality of FGF versus TGF-ß signaling in distinct CAF populations that promote cancer development through modulation of different processes.

Androgen receptor functions as transcriptional repressor of cancer-associated fibroblast activation.

The aging-associated increase of cancer risk is linked with stromal fibroblast senescence and concomitant cancer-associated fibroblast (CAF) activation. Surprisingly little is known about the role of androgen receptor (AR) signaling in this context. We have found downmodulated AR expression in dermal fibroblasts underlying premalignant skin cancer lesions (actinic keratoses and dysplastic nevi) as well as in CAFs from the 3 major skin cancer types, squamous cell carcinomas (SCCs), basal cell carcinomas, and melanomas. Functionally, decreased AR expression in primary human dermal fibroblasts (HDFs) from multiple individuals induced early steps of CAF activation, and in an orthotopic skin cancer model, AR loss in HDFs enhanced tumorigenicity of SCC and melanoma cells. Forming a complex, AR converged with CSL/RBP-Jκ in transcriptional repression of key CAF effector genes. AR and CSL were positive determinants of each other's expression, with BET inhibitors, which counteract the effects of decreased CSL, restoring AR expression and activity in CAFs. Increased AR expression in these cells overcame the consequences of CSL loss and was by itself sufficient to block the growth and tumor-enhancing effects of CAFs on neighboring cancer cells. As such, the findings establish AR as a target for stroma-focused cancer chemoprevention and treatment.

Autophagy Controls CSL/RBPJκ Stability through a p62/SQSTM1-Dependent Mechanism.

Cancer-associated fibroblasts (CAFs) are important at all tumor stages. CSL/RBPJκ suppresses the gene expression program leading to CAF activation and associated metabolic reprogramming, as well as autophagy. Little is known about CSL protein turnover, especially in the tumor microenvironment. We report that, in human dermal fibroblasts (HDFs), conditions inducing autophagy-often found in tumor stroma-down-regulate CSL protein levels but do not affect its mRNA levels. Genetic or pharmacologic targeting of the autophagic machinery blocks CSL down-modulation. Mechanistically, endogenous CSL associates with the autophagy and signaling adaptor p62/SQSTM1, which is required for CSL down-modulation by autophagy. This is functionally significant, because both CSL and p62 levels are lower in skin cancer-derived CAFs, in which autophagy is increased. Increasing cellular CSL levels stabilizes p62 and down-modulates the autophagic process. We reveal here an autophagy-initiated mechanism for CSL down-modulation, which could be targeted for stroma-focused cancer prevention and treatment.

Collagen disruption as a marker for basal cell carcinoma in presurgical margin detection.

INTRODUCTION AND OBJECTIVE: Nonmelanoma skin cancers (NMSCs) are the most common malignancies in the United States. Surgery is the most common treatment for these tumors, but pre-operative identification of surgical margins is challenging. The objective in this study was to determine whether optical polarization imaging (OPI) could be used prior to surgery to detect the extent of subclinical tumor spread by monitoring disruption in collagen. MATERIALS AND METHODS: OPI is a non-invasive and rapid imaging modality that highlights the structure of dermal collagen. OPI was used preoperatively at wavelengths of 440 and 640 nm to perform imaging of NMSCs on six patients scheduled to undergo Mohs surgery for biopsy-proven basal cell carcinoma. This pilot study did not alter the course of routine MMS for any of the patients. The surgeon was blinded from the preoperative imaging results and completed the entire procedure without relying on the new technology. The study was conducted in an outpatient surgical setting. Patients over 18 years of age with biopsy-proven basal cell carcinoma participated. RESULTS AND CONCLUSION: OPI accurately predicted the presence or absence of tumor at the surgical margin in six out of six cases, as confirmed on histology. OPI may allow efficient surgical planning by identifying tumor extension beyond visibly involved skin. Lasers Surg. Med. 50:902-907, 2018. © 2018 Wiley Periodicals, Inc.

The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level.

The integrity of stratified epithelia depends on the ability of progenitor cells to maintain a balance between proliferation and differentiation. While much is known about the transcriptional pathways underlying progenitor cells' behavior in the epidermis, the role of posttranscriptional regulation by mRNA binding proteins-a rate-limiting step in sculpting the proteome-remains poorly understood. Here we report that the RNA binding protein YBX1 (Y-box binding protein-1) is a critical effector of progenitors' function in the epidermis. YBX1 expression is restricted to the cycling keratinocyte progenitors in vivo and its genetic ablation leads to defects in the architecture of the skin. We further demonstrate that YBX1 negatively controls epidermal progenitor senescence by regulating the translation of a senescence-associated subset of cytokine mRNAs via their 3' untranslated regions. Our study establishes YBX1 as a posttranscriptional effector required for maintenance of epidermal homeostasis.

The ULK3 Kinase Is Critical for Convergent Control of Cancer-Associated Fibroblast Activation by CSL and GLI.

The connection between signaling pathways activating cancer-associated fibroblasts (CAFs) remains to be determined. Metabolic alterations linked to autophagy have also been implicated in CAF activation. CSL/RBPJ, a transcriptional repressor that mediates Notch signaling, suppresses the gene expression program(s), leading to stromal senescence and CAF activation. Deregulated GLI signaling can also contribute to CAF conversion. Here, we report that compromised CSL function depends on GLI activation for conversion of human dermal fibroblasts into CAFs, separately from cellular senescence. Decreased CSL upregulates the expression of the ULK3 kinase, which binds and activates GLI2. Increased ULK3 also induces autophagy, which is unlinked from GLI and CAF activation. ULK3 upregulation occurs in the CAFs of several tumor types, and ULK3 silencing suppresses the tumor-enhancing properties of these cells. Thus, ULK3 links two key signaling pathways involved in CAF conversion and is an attractive target for stroma-focused anti-cancer intervention.

Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation.

Cancer-associated fibroblasts (CAFs) are important for tumor initiation and promotion. CSL, a transcriptional repressor and Notch mediator, suppresses CAF activation. Like CSL, ATF3, a stress-responsive transcriptional repressor, is down-modulated in skin cancer stromal cells, and Atf3 knockout mice develop aggressive chemically induced skin tumors with enhanced CAF activation. Even at low basal levels, ATF3 converges with CSL in global chromatin control, binding to few genomic sites at a large distance from target genes. Consistent with this mode of regulation, deletion of one such site 2 Mb upstream of IL6 induces expression of the gene. Observed changes are of translational significance, as bromodomain and extra-terminal (BET) inhibitors, unlinking activated chromatin from basic transcription, counteract the effects of ATF3 or CSL loss on global gene expression and suppress CAF tumor-promoting properties in an in vivo model of squamous cancer-stromal cell expansion. Thus, ATF3 converges with CSL in negative control of CAF activation with epigenetic changes amenable to cancer- and stroma-focused intervention.