Protein kinase PKN1 represses Wnt/ß-catenin signaling in human melanoma cells.

Advances in phosphoproteomics have made it possible to monitor changes in protein phosphorylation that occur at different steps in signal transduction and have aided the identification of new pathway components. In the present study, we applied this technology to advance our understanding of the responses of melanoma cells to signaling initiated by the secreted ligand WNT3A. We started by comparing the phosphopeptide patterns of cells treated with WNT3A for different periods of time. Next, we integrated these data sets with the results from a siRNA screen that targeted protein kinases. This integration of siRNA screening and proteomics enabled us to identify four kinases that exhibit altered phosphorylation in response to WNT3A and that regulate a luciferase reporter of ß-catenin-responsive transcription (ß-catenin-activated reporter). We focused on one of these kinases, an atypical PKC kinase, protein kinase N1 (PKN1). Reducing the levels of PKN1 with siRNAs significantly enhances activation of ß-catenin-activated reporter and increases apoptosis in melanoma cell lines. Using affinity purification followed by mass spectrometry, we then found that PKN1 is present in a protein complex with a WNT3A receptor, Frizzled 7, as well as with proteins that co-purify with Frizzled 7. These data establish that the protein kinase PKN1 inhibits Wnt/ß-catenin signaling and sensitizes melanoma cells to cell death stimulated by WNT3A.

Dysregulation of Wnt/ß-catenin signaling in gastrointestinal cancers.

Aberrant Wnt/ß-catenin signaling is widely implicated in numerous malignancies, including cancers of the gastrointestinal tract. Dysregulation of signaling is traditionally attributed to mutations in Axin, adenomatous polyposis coli, and ß-catenin that lead to constitutive hyperactivation of the pathway. However, Wnt/ß-catenin signaling is also modulated through various other mechanisms in cancer, including cross talk with other altered signaling pathways. A more complex view of Wnt/ß-catenin signaling and its role in gastrointestinal cancers is now emerging as divergent phenotypic outcomes are found to be dictated by temporospatial context and relative levels of pathway activation. This review summarizes the dysregulation of Wnt/ß-catenin signaling in colorectal carcinoma, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma, with particular emphasis on the latter two. We conclude by addressing some of the major challenges faced in attempting to target the pathway in the clinic.

Activated Wnt/beta-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model.

This study demonstrates that in malignant melanoma, elevated levels of nuclear beta-catenin in both primary tumors and metastases correlate with reduced expression of a marker of proliferation and with improved survival, in contrast to colorectal cancer. The reduction in proliferation observed in vivo is recapitulated in B16 murine melanoma cells and in human melanoma cell lines cultured in vitro with either WNT3A or small-molecule activators of beta-catenin signaling. Consistent with these results, B16 melanoma cells expressing WNT3A also exhibit decreased tumor size and decreased metastasis when implanted into mice. Genome-wide transcriptional profiling reveals that WNT3A up-regulates genes implicated in melanocyte differentiation, several of which are down-regulated with melanoma progression. These findings suggest that WNT3A can mediate transcriptional changes in melanoma cells in a manner reminiscent of the known role of Wnt/beta-catenin signaling in normal melanocyte development, thereby altering melanoma cell fate to one that may be less proliferative and potentially less aggressive. Our results may explain the observed loss of nuclear beta-catenin with melanoma progression in human tumors, which could reflect a dysregulation of cellular differentiation through a loss of homeostatic Wnt/beta-catenin signaling.

Optimizing Narrowband UVB Phototherapy: Is It More Challenging for Your Older Patients?

Narrowband UVB (NB-UVB) phototherapy remains versatile, safe, and efficacious for multiple dermatologic conditions even with recent pharmacologic treatment advances. Polypharmacy contributes to prescribers pursuing phototherapy as a nonpharmacologic treatment, but some wonder if it is as effective and safe for older patients. This study aimed to determine if NB-UVB is equally effective in both older and younger adults treated with the same protocol and to examine the association between photosensitizing medications, clearance, and erythema rates in older vs younger adults.

Phototherapy: Safe and Effective for Challenging Skin Conditions in Older Adults.

Identifying safe, effective, and affordable evidence-based dermatologic treatments for older adults can be challenging because of age-related changes. Few studies have examined the effectiveness of phototherapy in older adults. Our retrospective study of patients 65 years and older who were treated with narrowband UVB(NB-UVB) phototherapy aimed to (1) identify the most common dermatologic conditions treated with phototherapy in older adults, (2) examine the effectiveness and safety of phototherapy in older adults, and (3) compare the outcomes to 2 similar studies in the United Kingdom and Turkey.

A re-evaluation of the "oncogenic" nature of Wnt/beta-catenin signaling in melanoma and other cancers.

In cancer, Wnt/beta-catenin signaling is ubiquitously referred to as an "oncogenic" pathway that promotes tumor progression. This review examines how the regulation and downstream effects of Wnt/beta-catenin signaling in cancer varies depending on cellular context, with a focus on malignant melanoma. We emphasize that the cellular homeostasis of Wnt/beta-catenin signaling may represent a more appropriate concept than the simplified view of the Wnt/beta-catenin pathway as either oncogenic or tumor-suppressing. Ultimately, a more refined understanding of the contextual regulation of Wnt/beta-catenin signaling will be essential for addressing if and how therapeutic targeting of this pathway could be leveraged for patient benefit.

Management of melanocytic lesions in the primary care setting.

As the incidence of melanoma continues to increase, the role of primary care physicians in managing suspect melanocytic lesions will become increasingly important. The management of suspect melanocytic lesions in the primary care setting can be challenging, and a missed diagnosis of melanoma can have devastating consequences. This review focuses on practical aspects of the evaluation of melanocytic lesions, including patient history, physical examination findings, and the natural history of nevi and melanoma. It also discusses melanocytic lesions that warrant special attention and outlines information that should be provided on the pathology requisition form. Finally, it addresses the potential difficulties in interpreting pathology reports and reviews the implications of pathology results for patient counseling, education, and follow-up.

Subungual Bowen disease in a patient with epidermodysplasia verruciformis presenting clinically as longitudinal melanonychia.

Epidermodysplasia verruciformis (EV) is a rare autosomal-recessive condition associated with a predisposition to infection with specific types of human papillomaviruses. A spectrum of wart-like lesions on the face, dorsa of the hands, and legs are characteristic clinical findings. Lesions usually develop in early childhood, persist, and may eventuate in cutaneous squamous cell carcinoma, usually in sun-exposed areas. These lesions are locally destructive and sometimes metastasize. We present a case of a 34-year-old African American woman with EV with a 9-month history of a right index finger ungual longitudinal pigmented band and nail splitting. Biopsy showed hyperkeratotic and parakeratotic subungual epithelium with verrucous hyperplasia. The superficial keratinocytes showed koilocytic changes. In addition, there was extensive, focally full-thickness keratinocyte dysmaturation with variable nuclear atypia and numerous mitotic figures, without apparent invasion. An associated melanocytic hyperplasia (confirmed by Melan-A stain), composed of large, pigment-laden dendritic melanocytes, was present without appreciable atypia or pagetoid spread. The findings are of a squamous cell carcinoma in situ arising in association with EV with incidental melanocytic hyperplasia. To the best of our knowledge, this is first report of a subungual presentation of this condition with associated melanonychia.

WNTS and WNT receptors as therapeutic tools and targets in human disease processes.

The body of scientific literature linking Wnts and Wnt-associated proteins to human disease processes continues to grow in parallel with new discoveries from basic science laboratories that further characterize the elaborate cellular events following the binding of Wnts to their receptors. While Wnt-mediated signaling has long been known to play a major role in human carcinogenesis, accumulating evidence indicates that Wnts are also important mediators of inflammation and recovery from injury. The binding of secreted Wnt ligands to their receptors offers an attractive and accessible target for therapeutic regulation of these signaling pathways. Several promising preliminary studies have already addressed potential avenues for the manipulation of Wnt signaling in disease processes. This review will focus on disease processes involving the regulation of Wnt signaling at the level of Wnt binding to its target receptors. Wnt proteins, Wnt receptors, and secreted Wnt inhibitors are attractive as potential therapeutic agents and targets due to their extracellular location. In addition, since Wnt signaling results in a diverse array of downstream intracellular events, many of which are not fully understood, the targeting of this pathway at the most upstream site of pathway activation also provides a strategic advantage for therapy. As the list of Wnt-related diseases continues to grow, advances in our understanding of the biochemical and molecular mechanisms underlying Wnt signaling may ultimately translate into innovative ways to treat Wnt-related disease processes in patients.

Winnie the Pooh and psoriasis too: an isomorphic response of guttate psoriasis in a tattoo.

Since Koebner's first observance of the isomorphic response of psoriasis in a tattoo in 1872, a paucity of written reports has appeared in the literature describing this phenomenon, although it appears to be common knowledge among dermatologists. We report a strep-induced guttate psoriasis isomorphic response in a Winnie the Pooh tattoo in a 19-year-old.

Association between Lithium Use and Melanoma Risk and Mortality: A Population-Based Study.

Laboratory studies show that lithium, an activator of the Wnt/ß-catenin signaling pathway, slows melanoma progression, but to our knowledge no published epidemiologic studies have explored this association. We conducted a retrospective cohort study of adult white Kaiser Permanente Northern California members (n = 2,213,848) from 1997-2012 to examine the association between lithium use and melanoma risk. Lithium exposure (n = 11,317) was assessed from pharmacy databases, serum lithium levels were obtained from electronic laboratory databases, and incident cutaneous melanomas (n = 14,056) were identified from an established cancer registry. In addition to examining melanoma incidence, melanoma hazard ratios and 95% confidence intervals for lithium exposure were estimated using Cox proportional hazards models, adjusted for potential confounders. Melanoma incidence per 100,000 person-years among lithium-exposed individuals was 67.4, compared with 92.5 in unexposed individuals (P = 0.027). Lithium-exposed individuals had a 32% lower risk of melanoma (hazard ratio = 0.68, 95% confidence interval = 0.51-0.90) in unadjusted analysis, but the estimate was attenuated and nonsignificant in adjusted analysis (adjusted hazard ratio = 0.77, 95% confidence interval = 0.58-1.02). No lithium-exposed individuals presented with thick (>4 mm) or advanced-stage melanoma at diagnosis. Among melanoma patients, lithium-exposed individuals were less likely to suffer melanoma-associated mortality (rate = 4.68/1,000 person-years) compared with the unexposed (rate = 7.21/1,000 person-years). Our findings suggest that lithium may reduce melanoma risk and associated mortality.

Hereditary woolly hair and keratosis pilaris.

We describe a family with woolly hair and ulerythema ophryogenes spanning four generations. Both woolly hair and ulerythema ophryogenes have been associated with Noonan syndrome and cardiofaciocutaneous syndrome (CFC), two disorders with considerable phenotypic overlap. This family did not exhibit any of the other findings characteristic of either Noonan syndrome or CFC, similar to a previously described pedigree with hereditary woolly hair. Woolly hair elicits a broad differential diagnosis, including woolly hair nevus and several genodermatoses. Our report reviews the evaluation of woolly hair and discusses the conditions associated with this physical finding.

Control of the interface between heterotypic cell populations reveals the mechanism of intercellular transfer of signaling proteins.

Direct intercellular transfer of cellular components is a recently described general mechanism of cell­cell communication. It is a more non-specific mode of intercellular communication that is not actively controlled by the participating cells. Though membrane bound proteins and small non-protein cytosolic components have been shown to be transferred between cells, the possibility of transfer of cytosolic proteins has not been clearly established, and its mechanism remains unexplained. Using a cell­cell pair of metastatic melanoma and endothelial cells, known to interact at various stages during cancer progression, we show that cytosolic proteins can indeed be transferred between heterotypic cells. Using precise relative cell patterning we provide evidence that this transfer depends on extent of the interface between heterotypic cell populations. This result is further supported by a mathematical model capturing various experimental conditions. We further demonstrate that cytosolic protein transfer can have important functional consequences for the tumor­stroma interactions, e.g., in heterotypic transfer of constitutively activated BRAF, a common melanoma associated mutation, leading to an enhanced activation of the downstream MAPK pathway. Our results suggest that cytosolic protein transfer can have important consequences for regulation of processes involving physical co-location of heterotypic cell types, particularly in invasive cancer growth.

Acute lymphoblastic leukemia presenting with urticarial plaques and hypereosinophilia in a child.

Our report describes a previously healthy 10-year-old female who was seen for urticarial plaques and mild loss of appetite. An initial laboratory workup revealed an elevated leukocyte count of 30,000/microL and a peripheral eosinophil count of 22,500/microL. A skin biopsy showed a marked hypersensitivity tissue response with abundant eosinophils. Further investigation of her peripheral eosinophilia uncovered Giardia lamblia in a stool sample. Despite treatment with the anti-parasitic agent furazolidone, the patient's urticarial plaques, leukocyte count, and peripheral eosinophil count remained unchanged. A bone marrow biopsy confirmed a diagnosis of acute lymphoblastic leukemia (ALL). ALL with hypereosinophilia (ALL/Eo) represents a rare and distinct subset of ALL, with more than 30 cases documented in the literature. Our discussion summarizes the clinical aspects of this disease and reviews the reported dermatological manifestations of ALL/Eo.

Targeted BRAF inhibition impacts survival in melanoma patients with high levels of Wnt/ß-catenin signaling.

Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas. We and others have previously observed an association of elevated nuclear ß-catenin with improved survival in molecularly-unselected melanoma patients. This study sought to determine whether levels of Wnt/ß-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi). We performed automated quantification of ß-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients. Unexpectedly, patients with higher nuclear ß-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi. In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/ß-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells. Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/ß-catenin signaling to affect patient outcomes. Studies with larger patient cohorts are required to determine whether nuclear ß-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi. Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.

WNT5A enhances resistance of melanoma cells to targeted BRAF inhibitors.

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF(V600E/K)) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.

Activation of Wnt/ß-catenin signaling increases apoptosis in melanoma cells treated with trail.

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/ß-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/ß-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/ß-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular ß-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/ß-catenin signaling, and suggest that strategies to enhance Wnt/ß-catenin signaling in combination with TRAIL agonists warrant further investigation.

FAM129B is a novel regulator of Wnt/ß-catenin signal transduction in melanoma cells.

The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/ß-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/ß-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of ß-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/ß-catenin signaling.  Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a ß-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/ß-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/ß-catenin signaling to apoptosis in melanoma.