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DNA damage and repair in human cells exposed to ultraviolet light (254 nm) or to psoralen derivatives plus 360 nm light were compared by means of a variety of analytic techniques. The two kinds of damage show considerable structural similarity; both involve cyclobutyl bonds to 5,6 positions of pyrimidines as major products and have various minor products. In purified DNA, pyrimidine dimers, but not psoralen adducts, cause structural distortions that are substances for digestion with single-strand-specific nucleases. Whereas pyrimidine dimers are randomly produced in chromatin, psoralen adducts, are concentrated approximately 2- to 4-fold in linker regions of chromatin at doses that are not highly lethal. Chromatin shows considerable mobility; assignment of DNA to linker or core regions is not permanent, and psoralen adducts initially concentrated in linker regions become randomized after 10 hr. Pyrimidine dimers and psoralen adducts are excised by normal cells but not by repair-deficient xeroderma pigmentosum cells. This repair process requires DNA polymerase alpha, but its rate in ultraviolet-damaged cells is twice that in psoralen-damaged cells. Conversion of monoadducts to DNA-DNA crosslinks reduces the rate of repair because of the increased complexity of the damaged site.

Therapy of BRAF -mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF -mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS - and NF1 -mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

Melanoma occurs as a consequence of inherited susceptibility to the disease and exposure to UV radiation (UVR) and is characterized by uncontrolled cellular proliferation and a high mutational load. The precise mechanisms by which UVR contributes to the development of melanoma remain poorly understood. Here we show that activation of nuclear receptor coactivator 3 (NCOA3) promotes melanomagenesis through regulation of UVR sensitivity, cell-cycle progression, and circumvention of the DNA damage response (DDR). Downregulation of NCOA3 expression, either by genetic silencing or small-molecule inhibition, significantly suppressed melanoma proliferation in melanoma cell lines and patient-derived xenografts. NCOA3 silencing suppressed expression of xeroderma pigmentosum C and increased melanoma cell sensitivity to UVR. Suppression of NCOA3 expression led to activation of DDR effectors and reduced expression of cyclin B1, resulting in G 2 -M arrest and mitotic catastrophe. A SNP in NCOA3 (T960T) reduced NCOA3 protein expression and was associated with decreased melanoma risk, given a significantly lower prevalence in a familial melanoma cohort than in a control cohort without cancer. Overexpression of wild-type NCOA3 promoted melanocyte survival following UVR and was accompanied by increased levels of UVR-induced DNA damage, both of which were attenuated by overexpression of NCOA3 (T960T). These results describe NCOA3-regulated pathways by which melanoma can develop, with germline NCOA3 polymorphisms enabling enhanced melanocyte survival in the setting of UVR exposure, despite an increased mutational burden. They also identify NCOA3 as a novel therapeutic target for melanoma. SIGNIFICANCE: This study explores NCOA3 as a regulator of the DDR and a therapeutic target in melanoma, where activation of NCOA3 contributes to melanoma development following exposure to ultraviolet light., (©2021 American Association for Cancer Research.)

Background: Mitotic rate is a strong, independent prognostic factor in patients with melanoma. However, incorporating it into the melanoma staging system has proved challenging., Methods: The prognostic impact of mitotic rate was assessed in a melanoma cohort comprising 5050 patients from 2 geographically distinct populations. Computer-generated cut points for mitotic rate were constructed to determine its impact on melanoma-associated survival using Kaplan-Meier and multivariate regression analyses. The impact of mitotic rate also was assessed in randomly split training and validation sets., Results: Mitotic rate had a nonlinear impact on survival, as evidenced by unequally spaced cut points. An index incorporating these cut points that was constructed from one population produced significantly more accurate predictions of survival in the other population than using the entire scale of mitotic rate. An index constructed from the combined cohort was found to be independently predictive of survival, with an impact comparable to that of ulceration. Optimal high-versus-low cut points for mitotic rate were generated separately for each T category (<2 mitoses/mm 2 vs ≥2 mitoses/mm 2 for T1 melanoma, <4 mitoses/mm 2 vs ≥4 mitoses/mm 2 for T2 melanoma, <6 mitoses/mm 2 vs ≥6/mitoses/mm 2 for T3 melanoma, and <7 mitoses/mm 2 vs ≥7 mitoses/mm 2 for T4 melanoma). Using Kaplan-Meier analysis, elevated mitotic rate was found to have an impact on survival comparable to that of ulceration within each T category. Application of the index for mitotic rate that was constructed from the training data set demonstrated an independent impact in the validation data set, with a significance similar to that of ulceration., Conclusions: The results of the current study demonstrated the comparable prognostic impact of mitotic rate and ulceration, providing support for its reincorporation into the T category., (© 2020 American Cancer Society.)

Cell division and organismal development are exquisitely orchestrated and regulated processes. The dysregulation of the molecular mechanisms underlying these processes may cause cancer, a consequence of cell-intrinsic and/or cell-extrinsic events. Cellular DNA can be damaged by spontaneous hydrolysis, reactive oxygen species, aberrant cellular metabolism or other perturbations that cause DNA damage. Moreover, several environmental factors may damage the DNA, alter cellular metabolism or affect the ability of cells to interact with their microenvironment. While some environmental factors are well established as carcinogens, there remains a large knowledge gap of others owing to the difficulty in identifying them because of the typically long interval between carcinogen exposure and cancer diagnosis. DNA damage increases in cells harbouring mutations that impair their ability to correctly repair the DNA. Tumour predisposition syndromes in which cancers arise at an accelerated rate and in different organs - the equivalent of a sensitized background - provide a unique opportunity to examine how gene-environment interactions influence cancer risk when the initiating genetic defect responsible for malignancy is known. Understanding the molecular processes that are altered by specific germline mutations, environmental exposures and related mechanisms that promote cancer will allow the design of novel and effective preventive and therapeutic strategies.

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion., Competing Interests: Competing interest statement: C.C. and J.D.L. are coauthors on a 2018 research article.

The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors., Competing Interests: The authors declare no conflict of interest.

Xeroderma pigmentosum (XP) patients who lack the main damage recognition protein for global genome repair (GGR), XPC, have greatly increased skin cancer rates and elevated mutation frequencies originating from unrepaired ultraviolet photoproducts in the nontranscribed regions of the genome and in nontranscribed strands of expressed genes. But they show no increased mutations in transcribed strands. In contrast, cancer is absent from Cockayne syndrome (CS) patients that have defective transcription coupled repair (TCR) despite severe photosensitivity, CS patients remarkably show no elevation of UV induced mutagenesis implying that defective TCR may be protective against mutagenesis and carcinogenesis. Mutation avoidance in CS is postulated to occur through arrested transcription that generates a tripled stranded R loop consisting of DNA double strands and a nascent mRNA strand. R loops result in S phase apoptosis or activation of ATM kinase that causes a delay in DNA replication until TCR, or transcript cleavage by TFIIS or RNAaseH, relieves the transcription block. Resumption of replication then occurs on repaired DNA without concomitant mutagenesis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality., Competing Interests: The authors declare no conflict of interest.

Cancer and neurodegeneration represent the extreme responses of growing and terminally differentiated cells to cellular and genomic damage. The damage recognition mechanisms of nucleotide excision repair, epitomized by xeroderma pigmentosum (XP), and Cockayne syndrome (CS), lie at these extremes. Patients with mutations in the DDB2 and XPC damage recognition steps of global genome repair exhibit almost exclusively actinic skin cancer. Patients with mutations in the RNA pol II cofactors CSA and CSB, that regulate transcription coupled repair, exhibit developmental and neurological symptoms, but not cancer. The absence of skin cancer despite increased photosensitivity in CS implies that the DNA repair deficiency is not associated with increased ultraviolet (UV)-induced mutagenesis, unlike DNA repair deficiency in XP that leads to high levels of UV-induced mutagenesis. One attempt to explain the pathology of CS is to attribute genomic damage to endogenously generated reactive oxygen species (ROS). We show that inhibition of complex I of the mitochondria generates increased ROS, above an already elevated level in CSB cells, but without nuclear DNA damage. CSB, but not CSA, quenches ROS liberated from complex I by rotenone. Extracellular signaling by N-methyl-D-aspartic acid in neurons, however, generates ROS enzymatically through oxidase that does lead to oxidative damage to nuclear DNA. The pathology of CS may therefore be caused by impaired oxidative phosphorylation or nuclear damage from neurotransmitters, but without damage-specific mutagenesis. Environ. Mol. Mutagen. 57:322-330, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Microphthalmia-associated transcription factor (MITF) plays a critical and complex role in melanocyte transformation. Although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are incompletely understood. Recent studies identified an oncogenic role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression, in part through activation of BCL2, a canonical target of MITF signaling. Analysis of the BPTF promoter identified a putative MITF-binding site, suggesting that MITF may regulate BPTF expression. Overexpression of MITF resulted in up-regulation of BPTF in a panel of melanoma and melanocyte cell lines. shRNA-mediated down-regulation of MITF in melanoma cells was accompanied by down-regulation of BPTF and BPTF-regulated genes (including BCL2) and resulted in reduced proliferative capacity of melanoma cells. The suppression of cell growth mediated by MITF silencing was rescued by overexpression of BPTF cDNA. Binding of MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of BPTF, as evidenced by increased luciferase activity driven by the BPTF promoter. These results indicate that BPTF transduces key prosurvival signals driven by MITF, further supporting its important role in promoting melanoma cell survival and progression.

Background: There is debate as to whether deep inguinal lymph nodes should be removed with the superficial or femoral lymph nodes during sentinel lymph node biopsy for lower extremity melanoma, when both superficial and deep inguinal lymph nodes are identified by preoperative lymphoscintigraphy. This study evaluated the lymphatic drainage patterns in lower extremity melanoma to determine whether certain patterns could be used to limit the level of node removal and define the extent of dissection., Methods: A retrospective outcomes review was performed of lower extremity melanoma patients with excision and sentinel lymph node biopsy from 1995 to 2010. Outcomes included location of sentinel lymph node drainage basins, sentinel lymph node-positivity, and disease-free and overall survival, with drainage patterns compared between above- and below-knee melanomas., Results: Of 499 patients with lower extremity melanoma having sentinel lymph node biopsy, 356 had below-the-knee and 143 had above-the-knee melanoma. For below-knee melanoma, the node-positivity rate was 23 percent (63 of 271) for superficial inguinal, 0 percent (zero of three) for deep inguinal, and 50 percent (one of two) for popliteal basins. For above-knee melanoma, the positivity rate was 21 percent (24 of 113) for superficial inguinal, 33 percent (one of three) for deep inguinal basins, and 0 percent (zero of zero) for popliteal basins. Importantly, no patients with a negative superficial inguinal sentinel lymph node had a positive deep inguinal sentinel lymph node on final pathologic evaluation [corrected]., Conclusions: A difference was noted in patterns of sentinel lymph node drainage from lower extremity melanoma below and above the knee. Biopsy for deep inguinal basins may be deferred if there is simultaneous drainage to the superficial inguinal basin by preoperative lymphoscintigraphy., Clinical Question/level of Evidence: Risk, II.

Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models.

Importance: Airline pilots and cabin crew are occupationally exposed to higher levels of cosmic and UV radiation than the general population, but their risk of developing melanoma is not yet established., Objective: To assess the risk of melanoma in pilots and airline crew., Data Sources: PubMed (1966 to October 30, 2013), Web of Science (1898 to January 27, 2014), and Scopus (1823 to January 27, 2014)., Study Selection: All studies were included that reported a standardized incidence ratio (SIR), standardized mortality ratio (SMR), or data on expected and observed cases of melanoma or death caused by melanoma that could be used to calculate an SIR or SMR in any flight-based occupation., Data Extraction and Synthesis: Primary random-effect meta-analyses were used to summarize SIR and SMR for melanoma in any flight-based occupation. Heterogeneity was assessed using the χ2 test and I2 statistic. To assess the potential bias of small studies, we used funnel plots, the Begg rank correlation test, and the Egger weighted linear regression test., Main Outcomes and Measures: Summary SIR and SMR of melanoma in pilots and cabin crew., Results: Of the 3527 citations retrieved, 19 studies were included, with more than 266 431 participants. The overall summary SIR of participants in any flight-based occupation was 2.21 (95% CI, 1.76-2.77; P < .001; 14 records). The summary SIR for pilots was 2.22 (95% CI, 1.67-2.93; P = .001; 12 records). The summary SIR for cabin crew was 2.09 (95% CI, 1.67-2.62; P = .45; 2 records). The overall summary SMR of participants in any flight-based occupation was 1.42 (95% CI, 0.89-2.26; P = .002; 6 records). The summary SMR for pilots was 1.83 (95% CI, 1.27-2.63, P = .33; 4 records). The summary SMR for cabin crew was 0.90 (95% CI, 0.80-1.01; P = .97; 2 records)., Conclusions and Relevance: Pilots and cabin crew have approximately twice the incidence of melanoma compared with the general population. Further research on mechanisms and optimal occupational protection is needed.

Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Cockayne syndrome (CS) is a human DNA repair-deficient disease that involves transcription coupled repair (TCR), in which three gene products, Cockayne syndrome A (CSA), Cockayne syndrome B (CSB), and ultraviolet stimulated scaffold protein A (UVSSA) cooperate in relieving RNA polymerase II arrest at damaged sites to permit repair of the template strand. Mutation of any of these three genes results in cells with increased sensitivity to UV light and defective TCR. Mutations in CSA or CSB are associated with severe neurological disease but mutations in UVSSA are for the most part only associated with increased photosensitivity. This difference raises questions about the relevance of TCR to neurological disease in CS. We find that CSB-mutated cells, but not UVSSA-deficient cells, have increased levels of intramitochondrial reactive oxygen species (ROS), especially when mitochondrial complex I is inhibited by rotenone. Increased ROS would result in oxidative damage to mitochondrial proteins, lipids, and DNA. CSB appears to behave as an electron scavenger in the mitochondria whose absence leads to increased oxidative stress. Mitochondrial ROS, however, did not cause detectable nuclear DNA damage even when base excision repair was blocked by an inhibitor of polyADP ribose polymerase. Neurodegeneration in Cockayne syndrome may therefore be associated with ROS-induced damage in the mitochondria, independent of nuclear TCR. An implication of our present results is that mitochondrial dysfunction involving ROS has a major impact on CS-B pathology, whereas nuclear TCR may have a minimal role.

Ulceration is an important prognostic factor in melanoma whose biologic basis is poorly understood. Here we assessed the prognostic impact of pleckstrin homology domain-interacting protein (PHIP) copy number and its relationship to ulceration. PHIP copy number was determined using fluorescence in situ hybridization (FISH) in a tissue microarray cohort of 238 melanomas. Elevated PHIP copy number was associated with significantly reduced distant metastasis-free survival (DMFS; P=0.01) and disease-specific survival (DSS; P=0.009) by Kaplan-Meier analyses. PHIP FISH scores were independently predictive of DMFS (P=0.03) and DSS (P=0.03). Increased PHIP copy number was an independent predictor of ulceration status (P=0.04). The combined impact of increased PHIP copy number and tumor vascularity on ulceration status was highly significant (P<0.0001). Stable suppression of PHIP in human melanoma cells resulted in significantly reduced glycolytic activity in vitro, with lower expression of lactate dehydrogenase 5, hypoxia-inducible factor 1 alpha subunit, and vascular endothelial growth factor, and was accompanied by reduced microvessel density in vivo. These results provide further support for PHIP as a molecular prognostic marker of melanoma, and reveal a significant linkage between PHIP levels and ulceration. Moreover, they suggest that ulceration may be driven by increased glycolysis and angiogenesis.

Background: Previous studies showed conflicting and inconsistent results regarding the effect of anatomic location of the melanoma on sentinel lymph node (SLN) positivity and/or survival. This study was conducted to evaluate and compare the effect of the anatomic locations of primary melanoma on long-term clinical outcomes., Methods: All consecutive cutaneous melanoma patients (n=2,079) who underwent selective SLN dissection (SLND) from 1993 to 2009 in a single academic tertiary-care medical center were included. SLN positive rate, disease-free survival (DFS), and overall survival (OS) were determined. Kaplan-Meier survival, univariate, and multivariate analyses were performed to determine predictive factors for SLN status, DFS, and OS., Results: Head and neck melanoma (HNM) had the lowest SLN-positive rate at 10.8% (16.8% for extremity and 19.3% for trunk; P=0.002) but had the worst 5-year DFS (P<0.0001) and 5-year OS (P<0.0001) compared with other sites. Tumor thickness (P<0.001), ulceration (P<0.001), HNM location (P=0.001), mitotic rate (P<0.001), and decreasing age (P<0.001) were independent predictive factors for SLN-positivity. HNM with T3 or T4 thickness had significantly lower SLN positive rate compared with other locations (P≤0.05). Also, on multivariate analysis, HNM location versus other anatomic sites was independently predictive of decreased DFS and OS (P<0.001). By Kaplan-Meier analysis, HNM was associated significantly with the worst DFS and OS., Conclusions: Primary melanoma anatomic location is an independent predictor of SLN status and survival. Although HNM has a decreased SLN-positivity rate, it shows a significantly increased risk of recurrence and death as compared with other sites.

Cockayne syndrome is an autosomal recessive disease that covers a wide range of symptoms, from mild photosensitivity to severe neonatal lethal disorder. The pathology of Cockayne syndrome may be caused by several mechanisms such as a DNA repair deficiency, transcription dysregulation, altered redox balance and mitochondrial dysfunction. Conceivably each of these mechanisms participates during a different stage in life of a Cockayne syndrome patient. Endogenous reactive oxygen is considered as an ultimate cause of DNA damage that contributes to Cockayne syndrome pathology. Here we demonstrate that mitochondrial reactive oxygen does not cause detectable nuclear DNA damage. This observation implies that a significant component of Cockayne syndrome pathology may be due to abnormal mitochondrial function independent of nuclear DNA damage. The source of nuclear DNA damage to central nervous system tissue most likely occurs from extrinsic neurotransmitter signaling., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

For primary melanoma, there is a delay between the initial skin biopsy and sentinel lymph node dissection, which may cause anxiety for the patient. The consequences of this delay on disease progression are unknown. The goal of this study was to determine whether delay time for sentinel node dissection from the initial cutaneous melanoma biopsy affects patient outcomes. A retrospective analysis of 492 patients with melanoma who underwent a sentinel node dissection between 1993 and 1999 was carried out. The endpoints assessed were sentinel node tumor status, recurrence, and mortality. Time to sentinel node dissection was compared between patients with positive and negative sentinel nodes. Long-term survival and recurrence were evaluated in relation to the time between the cutaneous biopsy and the sentinel node dissection (delay time), comparing less than 40 days with at least 40 days. In total, 15.9% of patients had positive sentinel nodes. The median follow-up was 11.7 years. Positive sentinel node patients had a median delay of 35 days between the primary melanoma biopsy and the sentinel node dissection compared with 41 days for negative sentinel node patients (P=0.5). Kaplan-Meier survival curves showed that a delay time of less than 40 days versus at least 40 days was not related to recurrence of melanoma (log-rank P=0.13) or overall survival (log-rank P=0.14). On multivariate analysis of age, thickness, ulceration, and sentinel node status, there was no difference in disease-free survival (P=0.58) or overall survival (P=0.53) between the less than 40 days and the at least 40 days groups. A modest delay in sentinel node dissection from the initial melanoma biopsy does not adversely affect sentinel node status, recurrence, nor survival.

Three teams have applied whole-exome and proteome methods to identify a new cofactor of human RNA polymerase II that is required for the recovery of transcription on damaged templates. The identification of this new factor raises questions about the causal relationships between molecular mechanisms of transcription regulation and excision repair and developmental and neurological disease and nonmalignant skin photosensitivity.

Cockayne syndrome (CS) is a rare autosomal recessive neurodegenerative disease that is associated with mutations in either of two transcription-coupled DNA repair genes, CSA or CSB. Mice with a targeted mutation in the Csb gene (Cs-b(m/m)) exhibit a milder phenotype compared with human patients with mutations in the orthologous CSB gene. Mice mutated in Csb were crossed with mice lacking Xpc (Xp-c(-/-)), the global genome repair gene, to enhance the pathological symptoms. These Cs-b(m/m).Xp-c(-/-) mice were normal at birth but exhibited progressive failure to thrive, whole-body wasting, and ataxia and died at approximately postnatal day 21. Characterization of Cs-b(m/m).Xp-c(-/-) brains at postnatal stages demonstrated widespread reduction of myelin basic protein (MBP) and myelin in the sensorimotor cortex, the stratum radiatum, the corpus callosum, and the anterior commissure. Quantification of individual axons by electron microscopy showed a reduction in both the number of myelinated axons and the average diameter of myelin surrounding the axons. There were no significant differences in proliferation or oligodendrocyte differentiation between Cs-b(m/m).Xp-c(-/-) and Cs-b(m/+).Xp-c(-/-) mice. Rather, Cs-b(m/m).Xp-c(-/-) oligodendrocytes were unable to generate sufficient MBP or to maintain the proper myelination during early development. Csb is a multifunctional protein regulating both repair and the transcriptional response to reactive oxygen through its interaction with histone acetylase p300 and the hypoxia-inducible factor (HIF)1 pathway. On the basis of our results, combined with that of others, we suggest that in Csb the transcriptional response predominates during early development, whereas a neurodegenerative response associated with repair deficits predominates in later life.

Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.

The phosphorylation of H2Ax on its S139 site, γH2Ax, is important for the assembly of repair complexes at DNA double strand breaks (DSBs). The formation and functional role of γH2Ax after other kinds of DNA damage, especially UV light, where DSBs are rare, is less clear. Following UV light in the UVB and UVC ranges, complex distributions of γH2Ax can be identified, quite unlike the discrete enumerable foci seen after ionizing radiation. Several distinct distributions of γH2Ax occur: a low level nuclear-wide distribution of γH2Ax occurs during nucleotide excision repair; irregular focal distributions occur at arrested replication forks; high intensity nuclear-wide γH2Ax occurs in association with S-phase apoptosis. The intensity and distributions of γH2Ax vary according to the activity of excision repair, bypass polymerase and apoptotic caspases. The frequency of DSBs at arrested replication forks is low but highly variable in different cell types, and probably caused by enzymatic action. Despite the prominence of S139 phosphorylation following UV damage, mutation of this site has no influence on the UV damage response indicating that γH2Ax is a biomarker but not a participant in the UV-DNA damage response., (© 2011 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ~75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.

Timely intervention for cancer requires knowledge of its earliest genetic aberrations. Sequencing of tumors and their metastases reveals numerous abnormalities occurring late in progression. A means to temporally order aberrations in a single cancer, rather than inferring them from serially acquired samples, would define changes preceding even clinically evident disease. We integrate DNA sequence and copy number information to reconstruct the order of abnormalities as individual tumors evolve for 2 separate cancer types. We detect vast, unreported expansion of simple mutations sharply demarcated by recombinative loss of the second copy of TP53 in cutaneous squamous cell carcinomas (cSCC) and serous ovarian adenocarcinomas, in the former surpassing 50 mutations per megabase. In cSCCs, we also report diverse secondary mutations in known and novel oncogenic pathways, illustrating how such expanded mutagenesis directly promotes malignant progression. These results reframe paradigms in which TP53 mutation is required later, to bypass senescence induced by driver oncogenes.

The phosphorylation of H2Ax on its S139 site, γH2Ax, is important during DNA double-strand repair and is considered necessary for assembly of repair complexes, but its functional role after other kinds of DNA damage is less clear. We have measured the survival of isogenic mouse cell lines with the H2Ax gene knocked out, and replaced with wild-type or mutant (S139A) H2Ax genes, exposed to a range of agents with varied mechanisms of DNA damage. Knockout and mutant cells were sensitive to γ-rays, etoposide, temozolamide, and endogenously generated reactive oxygen species, each of which can include double-strand breaks among their spectra of DNA lesions. The absence or mutation of H2Ax had no influence on sensitivity to cisplatin or mitomycin C. Although UV light induced the highest levels of γH2Ax, mutation of S139 had no influence on UV sensitivity or the UV DNA damage response. Complete loss of H2Ax reduced the survival of cells exposed to UV light and reduced pChk1 induction, suggesting that sites other than S139 may impact the ATR-pChk1 pathway. The relative intensity of γH2Ax measured in Western blots in wild-type cells did not correlate with the functional importance of γH2Ax. The use of γH2Ax as a general biomarker of DNA damage is therefore potentially misleading because it is not an unambiguous indicator of double-strand breaks, and a significant fraction of DNA repair, especially involving nucleotide excision or crosslink repair, can occur without functional involvement of γH2Ax.

XPC, the main damage-recognition protein responsible for nucleotide excision repair of UVB damage to DNA, is lost or mutated in xeroderma pigmentosum group C (XP-C), a rare inherited disease characterized by high incidence and early onset of non-melanoma and melanoma skin cancers. The high incidence of skin cancers in XP-C patients suggests that loss of expression of XPC protein might also provide a selective advantage for initiation and progression of similar cancers in non XP-C patients in the general population. To test whether XPC is selectively lost in squamous cell carcinomas from non XP-C patients, we examined XPC expression by immunohistochemistry on a tissue microarray with 244 tissue cores, including in situ and invasive squamous-cell carcinomas (SCCs), keratoacanthoma (KA), and normal skin samples from both immunocompetent and immunosuppressed patients. We found that XPC expression was lost in 49% of invasive squamous cell carcinomas from immunocompetent patients and 59% from immunosuppressed patients. Loss of expression was correlated with deletions of chromosomal 3p and mutations in the XPC gene. The XPC gene is consequently inactivated or lost in almost half of squamous cell carcinomas from non XP-C patients. Loss or mutation of XPC may be an early event during skin carcinogenesis that provides a selective advantage for initiation and progression of squamous cell carcinomas in non XP-C patients.

UV irradiation induces histone variant H2AX phosphorylated on serine 139 (gammaH2AX) foci and high levels of pan-nuclear gammaH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of gammaH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of gammaH2AX response: a minority of gammaH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of gammaH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear gammaH2AX without foci of either gammaH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear gammaH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear gammaH2AX were further increased by loss of the bypass polymerase Pol eta and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damage-binding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced gammaH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear gammaH2AX, a preapoptotic signal associated with ATM- and JNK-dependent apoptosis during replication. The formation of gammaH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.

Xeroderma pigmentosum (XP) is a group of rare inherited human neurocutaneous diseases, and the group C (XPC) is the major group of patients with XP in Europe, North America, and South America. Current molecular diagnostic methods for XP require specialized, expensive, and time-consuming UV sensitivity and DNA repair assays followed by gene sequencing. To determine whether immunohistochemistry (IHC) would be a robust alternative method to diagnose patients with XPC, we stained sections of paraffin-embedded skin biopsies for XPC by IHC, using 69 archived blocks from confirmed or clinically suspect patients with XPA, XPC, XPD, XPE, and without XP. We found that XPC expression was strong in all skin biopsies from patients without (14 of 14) and other patients with XP (4 of 4), whereas XPC expression was lost in all biopsies from confirmed XPC patients (29 of 29). Patches of strong XPC signal could be detected in sun-damaged skin, squamous and basal cell carcinomas from patients with XPC that colocalized with strong expression of p53 and Ki-67. Patients with XPC can therefore be diagnosed by IHC from paraffin-embedded skin biopsies from regions of skin that are without sun damage or sun-induced tumors. IHC is therefore a robust alternative method to diagnose patients with XPC. This fast and inexpensive method should increase the options for the diagnosis of patients with XPC from paraffin-embedded skin biopsies and could be developed for other complementation groups.

Mutations in genes on the nucleotide excision repair pathway are associated with diseases, such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, that involve skin cancer and developmental and neurological symptoms. These mutations cause the defective repair of damaged DNA and increased transcription arrest but, except for skin cancer, the links between repair and disease have not been obvious. Widely different clinical syndromes seem to result from mutations in the same gene, even when the mutations result in complete loss of function. The mapping of mutations in recently solved protein structures has begun to clarify the links between the molecular defects and phenotypes, but the identification of additional sources of clinical variability is still necessary.

DNA strand breaks trigger marked phosphorylation of histone H2AX (i.e. gamma-H2AX). While DNA double-strand breaks (DSBs) provide a strong stimulus for this event, the accompanying structural alterations in chromatin may represent the actual signal that elicits gamma-H2AX. Our data show that changes in chromatin structure are sufficient to elicit extensive gamma-H2AX formation in the relative absence of DNA strand breaks. Cells subjected to hypotonic (0.05 M) treatment exhibit gamma-H2AX levels that are equivalent to those found after the induction of 80-200 DNA DSBs (i.e. 2-5 Gy). Despite this significant increase in phosphorylation, cell survival remains relatively unaffected (<10% cytotoxicity), and there is no significant increase in apoptosis. Nuclear staining profiles indicate that gamma-H2AX-positive cells induced under altered tonicity exhibit variable levels of staining, ranging from uniform pan staining to discrete punctate foci more characteristic of DNA strand breakage. The capability to induce significant gamma-H2AX formation under altered tonicity in the relative absence of DNA strand breaks suggests that this histone modification evolved in response to changes in chromatin structure.

Damaged DNA-binding protein (DDB), consisting of DDB1 and DDB2 subunits recognizes a wide spectrum of DNA lesions. DDB is dispensable for in vitro nucleotide excision repair (NER) reaction, but stimulates this reaction especially for cyclobutane pyrimidine dimer (CPD). Here we show that DDB directly interacts with XPA, one of core NER factors, mainly through DDB2 subunit and the amino-acid residues between 185 and 226 in XPA are important for the interaction. Interestingly, the point mutation causing the substitution from Arg-207 to Gly, which was previously identified in a XP-A revertant cell-line XP129, diminished the interaction with DDB in vitro and in vivo. In a defined system containing R207G mutant XPA and other core NER factors, DDB failed to stimulate the excision of CPD, although the mutant XPA was competent for the basal NER reaction. Moreover, in vivo experiments revealed that the mutant XPA is recruited to damaged DNA sites with much less efficiency compared with wild-type XPA and fails to support the enhancement of CPD repair by ectopic expression of DDB2 in SV40-transformed human cells. These results suggest that the physical interaction between DDB and XPA plays an important role in the DDB-mediated NER reaction.

Human DNA polymerase eta (pol eta) can replicate across UV-induced pyrimidine dimers, and defects in the gene encoding pol eta result in a syndrome called xeroderma pigmentosum variant (XP-V). XP-V patients are prone to the development of cancer in sun-exposed areas, and cells derived from XP-V patients demonstrate increased sensitivity to UV radiation and a higher mutation rate compared with wild-type cells. pol eta has been shown to replicate across a wide spectrum of DNA lesions introduced by environmental or chemotherapeutic agents, or during nucleotide starvation, suggesting that the biological roles for pol eta are not limited to repair of UV-damaged DNA. The high error rate of pol eta requires that its intracellular activity be tightly regulated. Here, we show that the phosphorylation of pol eta increased after UV irradiation, and that treatment with caffeine, siRNA against ATR, or an inhibitor of PKC (calphostin C), reduced the accumulation of pol eta at stalled replication forks after UV irradiation or treatment with cisplatin and gemcitabine. Site-specific mutagenesis (S587A and T617A) of pol eta at two putative PKC phosphorylation sites located in the protein-protein interaction domain prevented nuclear foci formation induced by UV irradiation or treatment with gemcitabine/cisplatin. In addition, XP-V cell lines stably expressing either the S587A or T617A mutant form of pol eta were more sensitive to UV radiation and gemcitabine/cisplatin than control cells expressing wild-type pol eta. These results suggest that phosphorylation is one mechanism by which the cellular activity of pol eta is regulated.

Cancer, aging, and neurodegeneration are all associated with DNA damage and repair in complex fashions. Aging appears to be a cell and tissue-wide process linked to the insulin-dependent pathway in several DNA repair deficient disorders, especially in mice. Cancer and neurodegeneration appear to have complementary relationships to DNA damage and repair. Cancer arises from surviving cells, or even stem cells, that have down-regulated many pathways, including apoptosis, that regulate genomic stability in a multi-step process. Neurodegeneration however occurs in nondividing neurons in which the persistence of apoptosis in response to reactive oxygen species is, itself, pathological. Questions that remain open concern: sources and chemical nature of naturally occurring DNA damaging agents, especially whether mitochondria are the true source; the target tissues for DNA damage and repair; do the human DNA repair deficient diseases delineate specific pathways of DNA damage relevant to clinical outcomes; if naturally occurring reactive oxygen species are pathological in human repair deficient disease, would anti-oxidants or anti-apoptotic agents be feasible therapeutic agent?

The discovery that xeroderma pigmentosum was a sun-sensitive hereditary human disease that was deficient in DNA repair was made when research into the fundamental mechanisms of nucleotide excision repair was in its infancy. The linkage between DNA damage, DNA repair and human cancer stimulated an enormous subsequent growth of the field of DNA repair and the identification of other repair deficient diseases and other repair pathways. This growth has established DNA repair as a central factor for maintaining genomic stability and preventing cancer, neurodegenerative disease and aging. The study of DNA repair impacts many other areas including human genetics, signal transduction, protein structure, DNA-protein interactions, DNA replication and recombination, transcription, telomere maintenance, development, differentiation, ecology and evolution.