An Investigation of Structure-Activity Relationships and Cell Death Mechanisms of the Marine Alkaloids Discorhabdins in Merkel Cell Carcinoma Cells.

A library of naturally occurring and semi-synthetic discorhabdins was assessed for their effects on Merkel cell carcinoma (MCC) cell viability. The set included five new natural products and semi-synthetic compounds whose structures were elucidated with NMR, HRMS, and ECD techniques. Several discorhabdins averaged sub-micromolar potency against the MCC cell lines tested and most of the active compounds showed selectivity towards virus-positive MCC cell lines. An investigation of structure-activity relationships resulted in an expanded understanding of the crucial structural features of the discorhabdin scaffold. Mechanistic cell death assays suggested that discorhabdins, unlike many other MCC-active small molecules, do not induce apoptosis, as shown by the lack of caspase activation, annexin V staining, and response to caspase inhibition. Similarly, discorhabdin treatment failed to increase MCC intracellular calcium and ROS levels. In contrast, the rapid loss of cellular reducing potential and mitochondrial membrane potential suggested that discorhabdins induce mitochondrial dysfunction leading to non-apoptotic cell death.

Differences in Merkel Cell Carcinoma Presentation and Outcomes Among Racial and Ethnic Groups.

Importance: Racial and ethnic differences in skin cancer outcomes are understudied. Delineating these differences in Merkel cell carcinoma (MCC) is needed to better understand this rare disease. Objective: To determine how MCC presentation and outcomes differ across racial and ethnic groups. Design, Setting, and Participants: This retrospective cohort study included patients diagnosed with MCC and followed up from 2000 through 2018 in the 18 population-based cancer registries of the National Cancer Institute's Surveillance, Epidemiology, and End Results Program. Patients without follow-up data were excluded. Data analysis occurred from March 12 to November 30, 2022. Main Outcomes and Measures: A Cox proportional hazards regression was conducted to determine associations between demographic variables (race and ethnicity, age, sex, and income) and clinical variables (stage at diagnosis, primary site, and diagnosis year) with MCC-specific survival. Results: Of the 9557 patients with MCC identified (6758 [70.7%] aged ≥70 years; 6008 [62.9%] male), 222 (2.3%) were Asian American or Pacific Islander, 146 (1.5%) Black, 541 (5.7%) Hispanic, and 8590 (89.9%) White. Hispanic patients had improved MCC-specific survival compared with White patients (hazard ratio, 0.82; 95% CI, 0.67-0.99; P = .04). Black patients had the lowest MCC-specific survival, but it was not statistically different from White patients (hazard ratio, 1.19; 95% CI, 0.86-1.60; P = .28). Hispanic and Black patients were less likely to present with a primary site of the head and neck than White patients (183 of 541 [33.8%] Hispanic patients and 45 of 146 [30.8%] Black patients vs 3736 of 8590 [43.5%] White patients; P < .001 and P = .002, respectively). Black patients presented more often than White patients with advanced disease at diagnosis (59 of 146 [40.4%] vs 2510 of 8590 [29.2%]; P = .004). Conclusions and Relevance: In this cohort study, there were differences between racial and ethnic groups in observed MCC outcomes and disease characteristics. Further investigations are warranted into the findings that, compared with White patients, Hispanic patients with MCC had improved outcomes and Black patients did not have worse outcomes despite presenting with more advanced disease.

Distinct Signatures of Genomic Copy Number Variants Define Subgroups of Merkel Cell Carcinoma Tumors.

Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated Merkel cell polyomavirus DNA (virus-positive MCC, VP-MCC) and carry a low somatic mutation burden whereas virus-negative MCC (VN-MCC) possess numerous ultraviolet-signature mutations. In contrast to viral oncogenes and sequence mutations, little is known about genomic structural variants in MCC. To identify copy number variants in commonly altered genes, we analyzed genomic DNA from 31 tumor samples using the Nanostring nCounter copy number cancer panel. Unsupervised clustering revealed three tumor groups with distinct genomic structural variant signatures. The first cluster was characterized by multiple recurrent deletions in genes such as RB1 and WT1. The second cluster contained eight VP-MCC and displayed very few structural variations. The final cluster contained one VP-MCC and four VN-MCC with predominantly genomic amplifications in genes like MDM4, SKP2, and KIT and deletions in TP53. Overall, VN-MCC contained more structure variation than VP-MCC but did not cluster separately from VP-MCC. The observation that most MCC tumors demonstrate a deletion-dominated structural group signature, independent of virus status, suggests a shared pathophysiology among most VP-MCC and VN-MCC tumors.

Identification of natural product modulators of Merkel cell carcinoma cell growth and survival.

Merkel cell carcinoma (MCC) is a rare, but aggressive skin cancer the incidence of which has increased significantly in recent years. The majority of MCCs have incorporated Merkel cell polyomavirus (VP-MCC) while the remainder are virus-negative (VN-MCC). Although a variety of therapeutic options have shown promise in treating MCC, there remains a need for additional therapeutics as well as probes for better understanding MCC. A high-throughput screening campaign was used to assess the ability of > 25,000 synthetic and natural product compounds as well as > 20,000 natural product extracts to affect growth and survival of VN-MCC and VP-MCC cell lines. Sixteen active compounds were identified that have mechanisms of action reported in the literature along with a number of compounds with unknown mechanisms. Screening results with pure compounds suggest a range of potential targets for MCC including DNA damage, inhibition of DNA or protein synthesis, reactive oxygen species, and proteasome inhibition as well as NFκB inhibition while also suggesting the importance of zinc and/or copper binding. Many of the active compounds, particularly some of the natural products, have multiple reported targets suggesting that this strategy might be a particularly fruitful approach. Processing of several active natural product extracts resulted in the identification of additional MCC-active compounds. Based on these results, further investigations focused on natural products sources, particularly of fungal origin, are expected to yield further potentially useful modulators of MCC.

ΔNp63α suppresses cells invasion by downregulating PKCγ/Rac1 signaling through miR-320a.

ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration, and invasion. ΔNp63α has been shown to regulate several microRNAs that are involved in development and cancer. We identified miRNA miR-320a as a positively regulated target of ΔNp63α. Previous studies have shown that miR-320a is downregulated in colorectal cancer and targets the small GTPase Rac1, leading to a reduction in noncanonical WNT signaling and EMT, thereby inhibiting tumor metastasis and invasion. We showed that miR-320a is a direct target of ΔNp63α. Knockdown of ΔNp63α in HaCaT and A431 cells downregulates miR-320a levels and leads to a corresponding elevation in PKCγ transcript and protein levels. Rac1 phosphorylation at Ser71 was increased in the absence of ΔNp63α, whereas overexpression of ΔNp63α reversed S71 phosphorylation of Rac1. Moreover, increased PKCγ levels, Rac1 phosphorylation and cell invasion observed upon knockdown of ΔNp63α was reversed by either overexpressing miR-320a mimic or Rac1 silencing. Finally, silencing PKCγ or treatment with the PKC inhibitor Gö6976 reversed increased Rac1 phosphorylation and cell invasion observed upon silencing ΔNp63α. Taken together, our data suggest that ΔNp63α positively regulates miR-320a, thereby inhibiting PKCγ expression, Rac1 phosphorylation, and cancer invasion.

Identification of novel ΔNp63α-regulated miRNAs using an optimized small RNA-Seq analysis pipeline.

Advances in high-throughput sequencing have enabled profiling of microRNAs (miRNAs), however, a consensus pipeline for sequencing of small RNAs has not been established. We built and optimized an analysis pipeline using Partek Flow, circumventing the need for analyzing data via scripting languages. Our analysis assessed the effect of alignment reference, normalization method, and statistical model choice on biological data. The pipeline was evaluated using sequencing data from HaCaT cells transfected with either a non-silencing control or siRNA against ΔNp63α, a p53 family member protein which is highly expressed in non-melanoma skin cancer and shown to regulate a number of miRNAs. We posit that 1) alignment and quantification to the miRBase reference provides the most robust quantitation of miRNAs, 2) normalizing sample reads via Trimmed Mean of M-values is the most robust method for accurate downstream analyses, and 3) use of the lognormal with shrinkage statistical model effectively identifies differentially expressed miRNAs. Using our pipeline, we identified previously unrecognized regulation of miRs-149-5p, 18a-5p, 19b-1-5p, 20a-5p, 590-5p, 744-5p and 93-5p by ΔNp63α. Regulation of these miRNAs was validated by RT-qPCR, substantiating our small RNA-Seq pipeline. Further analysis of these miRNAs may provide insight into ΔNp63α's role in cancer progression. By defining the optimal alignment reference, normalization method, and statistical model for analysis of miRNA sequencing data, we have established an analysis pipeline that may be carried out in Partek Flow or at the command line. In this manner, our pipeline circumvents some of the major hurdles encountered during small RNA-Seq analysis.

Modulation of miRNA-155 alters manganese nanoparticle-induced inflammatory response.

Regulation of gene expression by non-coding RNAs, such as microRNAs (miRNAs), is increasingly being examined in a variety of disciplines. Here we evaluated changes in miRNA expression following metallic nanoparticle (NP) exposure in a mouse neuronal co-culture model. Exposure to manganese (Mn) NPs resulted in oxidative stress, inflammation, and toxicity. Next-generation sequencing (NGS) following an 8 h exposure to Mn NPs (low and high doses) revealed several miRNA candidates that modulate NP induced responses. The lead candidate identified was miR-155, which showed a dose dependent decrease in expression upon Mn exposure. Introduction of a miR-155 mimic into the co-culture to restore miR-155 expression completely abrogated the Mn NP-induced gene and protein expression of inflammatory markers TNF-α and IL-6. Taken together, this study is the first report where global NP-induced miRNA expression changes were used to identify and then modulate negative impacts of metallic NP exposure in a neuronal model. These findings demonstrate that unique miRNA expression profiles provide novel targets for manipulating gene and protein expression, and therefore provide the potential of modifying cellular responses to NP exposure.

Role of vitamin D3 in modulation of ΔNp63α expression during UVB induced tumor formation in SKH-1 mice.

ΔNp63α, a proto-oncogene, is up-regulated in non-melanoma skin cancers and directly regulates the expression of both Vitamin D receptor (VDR) and phosphatase and tensin homologue deleted on chromosome ten (PTEN). Since ΔNp63α has been shown to inhibit cell invasion via regulation of VDR, we wanted to determine whether dietary Vitamin D3 protected against UVB induced tumor formation in SKH-1 mice, a model for squamous cell carcinoma development. We examined whether there was a correlation between dietary Vitamin D3 and ΔNp63α, VDR or PTEN expression in vivo in SKH-1 mice chronically exposed to UVB radiation and fed chow containing increasing concentrations of dietary Vitamin D3. Although we observed differential effects of the Vitamin D3 diet on ΔNp63α and VDR expression in chronically irradiated normal mouse skin as well as UVB induced tumors, Vitamin D3 had little effect on PTEN expression in vivo. While low-grade papillomas in mice exposed to UV and fed normal chow displayed increased levels of ΔNp63α, expression of both ΔNp63α and VDR was reduced in invasive tumors. Interestingly, in mice fed high Vitamin D3 chow, elevated levels of ΔNp63α were observed in both local and invasive tumors but not in normal skin suggesting that oral supplementation with Vitamin D3 may increase the proliferative potential of skin tumors by increasing ΔNp63α levels.

ΔNp63α represses nuclear translocation of PTEN by inhibition of NEDD4-1 in keratinocytes.

ΔNp63α maintains the proliferative potential of keratinocytes by inhibiting the transcription and nuclear localization of the tumor suppressor PTEN as shown earlier by our laboratory. The goal of this study was to define the mechanisms by which ΔNp63α mediates the nuclear exclusion of PTEN. We demonstrate here that ΔNp63α reduces the ubiquitination of PTEN, a key signaling event in the nuclear translocation of PTEN. The decrease in ubiquitinated PTEN correlated with the ability of ΔNp63α to bind to neuronal precursor developmentally down regulated 4 (NEDD4) promoter and transcriptionally repress the E3 ubiquitin ligase NEDD4-1. Knockdown of NEDD4-1 in cultured keratinocytes was sufficient to attenuate the increase in nuclear PTEN observed upon silencing of ΔNp63α. In vivo examination of normal skin demonstrated that ΔNp63α and NEDD4-1 were both expressed in the basal layer of the epidermis and this correlated with nuclear exclusion of PTEN. Altogether, these studies suggest that ΔNp63α-mediated suppression of nuclear PTEN in basal layer keratinocytes occurs through repression of NEDD4-1.

The PTEN-Akt pathway impacts the integrity and composition of mitotic centrosomes.

Loss of the tumor suppressor PTEN is observed in many human cancers that display increased chromosome instability and aneuploidy. The subcellular fractions of PTEN are associated with different functions that regulate cell growth, invasion and chromosome stability. In this study, we show a novel role for PTEN in regulating mitotic centrosomes. PTEN localization at mitotic centrosomes peaks between prophase and metaphase, paralleling the centrosomal localization of PLK-1 and γ-tubulin and coinciding with the time frame of centrosome maturation. In primary keratinocytes, knockdown of PTEN increased whole-cell levels of γ-tubulin and PLK-1 in an Akt-dependent manner and had little effect on recruitment of either protein to mitotic centrosomes. Conversely, knockdown of PTEN reduced centrosomal levels of pericentrin in an Akt-independent manner. Inhibition of Akt activation with MK2206 reduced the whole-cell and centrosome levels of PLK-1 and γ-tubulin and also prevented the recruitment of PTEN to mitotic centrosomes. This reduction in centrosome-associated proteins upon inhibition of Akt activity may contribute to the increase in defects in centrosome number and separation observed in metaphase cells. Concomitant PTEN knockdown and Akt inhibition reduced the frequency of metaphase cells with centrosome defects when compared with MK2206 treatment alone, indicating that both PTEN and pAkt are required to properly regulate centrosome composition during mitosis. The findings presented in this study demonstrate a novel role for PTEN and Akt in controlling centrosome composition and integrity during mitosis and provide insight into how PTEN functions as a multifaceted tumor suppressor.