RIPK4 downregulation impairs Wnt3A-stimulated invasiveness via Wnt/β\beta-catenin signaling in melanoma cells and tumor growth in vivo

Purpose The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/$\beta$-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood. Methods We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active $\beta$-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated. Results Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of $Ki67^{+}$ cells as well as reduced necrotic area and decreased levels of active $\beta$-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and $\beta$-catenin, as manifested by a decrease in the transcriptional activity of $\beta$-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on $\beta$-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls. Conclusion RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy

Deciphering the functional role of RIPK4 in melanoma

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-$1\beta$ level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-$\kappa$ signaling in a RIPK4-dependent ($RIPK4^{high}$) or independent ($RIPK4^{low}$) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial)

The effect of RORa expression on the development of biological malignancy of urinary bladder cancer

Background: Morbidity and mortality relating to urinary bladder cancer have remained largely unchanged for many years. Similarly, the five-year survival rate in this disease has not improved considerably. New developments in individualized therapy necessitate the search for novel factors that could predict the development of malignancy in UBC. In this study, we provide the first evidence that the expression of ROR alpha transcription factor influences the development of malignancy in UBC. Materials and methods: 105 patients with stage pT1-pT4 urothelial bladder carcinoma who underwent cystectomy were included in the study. 4 μm tissue samples were stained immunohistochemically with a polyclonal anti-RORa antibody. The expression of RORa by the tumor cells (TCs) was assessed by counting TCs with a cytoplasmic and/or nuclear staining for RORa per 1000 TCs. The association between the extent of RORa expression and non-classic differentiation, tumor advancement (pT), grade (G) and regional lymph node spread was analyzed. Results: The cytoplasmic expression of RORa was detected in near all analyzed tumor samples (104/105). The extent of RORa expression was significantly higher in tumors which were more malignant with more propensity for non-classic differentiation and lymph node metastasis. We noted a lower percentage of TCs expressing RORa in poorly differentiated tumors (G3), compared to tumors moderately and higher differentiated (G1/G2). Conclusions: Our results suggest that RORa may play a significant role in the progression of urinary bladder cancer. RORa has a broad spectrum of regulatory activity relating to cell and tissue differentiation the mechanism of which is not fully understood. This study represents another step in the process of understanding the mechanisms of RORa regulation and highlights its potential role as a therapeutic target in urothelial bladder cancer

Expression of PD-L1 in tumor and immune system cells affects the survival of patients with urinary bladder cancer

Background: The prediction of tumor malignancy is still one of the most demanding diagnostic tasksin urinary bladder cancer because of its clinicopathological heterogeneity. The aim of this study was toevaluate the expression of PD-L1 in tumor cells (TCs) and immune effector cells (IECs) as well as thepattern of distribution of PD-L1+ IECs within the tumor (dispersed or aggregated) and their associationwith survival of patients with pT1-pT4 urinary bladder cancer.Materials and methods: 110 patients with stage pT1-pT4 urothelial bladder carcinoma who underwentradical cystectomy/cystoprostatectomy between 2011 and 2014 were included in the study. Paraffin blocksmost representative of the tumor were selected for H&E staining as well as immunostaining with the useof rabbit anti-PD-L1 (Ventana clone SP142, Roche). In each sample, the area of the tumor containing PDL1+IECs, as well as, the pattern of distribution (dispersed or aggregated) of PD-L1+ immune effectorcells within the tumor were analyzed. In addition, the expression of PD-L1 in TCs was also assessed.Results: Patients had a shorter survival time in pT2-pT4 cases without TCs expressing PD-L1 (p = 0.007)and/or when PD-L1+ IECs displayed a predominantly dispersed pattern of distribution (p = 0.013).Conclusions: The expression of PD-L1 on TCs and IECs is a prognostic factor which allows for stratificationof patient survival in UBC. The predominance of dispersed or aggregated pattern of distribution ofPD-L1+ IECs in the tumor may be considered as a new prognostic factor in pT1-T4 UBC and indicate thefunctional status of the immune system

Vemurafenib and dabrafenib downregulates RIPK4 level

Vemurafenib and dabrafenib are BRAF kinase inhibitors (BRAFi) used for the treatment of patients with melanoma carrying the V600E BRAF mutation. However, melanoma cells develop resistance to both drugs when used as monotherapy. Therefore, mechanisms of drug resistance are investigated, and new molecular targets are sought that could completely inhibit melanoma progression. Since receptor-interacting protein kinase (RIPK4) probably functions as an oncogene in melanoma and its structure is similar to the BRAF protein, we analyzed the impact of vemurafenib and dabrafenib on RIPK4 in melanomas. The in silico study confirmed the high similarity of BRAF kinase domains to the RIPK4 protein at both the sequence and structural levels and suggests that BRAFi could directly bind to RIPK4 even more strongly than to ATP. Furthermore, BRAFi inhibited ERK1/2 activity and lowered RIPK4 protein levels in BRAF-mutated melanoma cells (A375 and WM266.4), while in wild-type BRAF cells (BLM and LoVo), both inhibitors decreased the level of RIPK4 and enhanced ERK1/2 activity. The phosphorylation of phosphatidylethanolamine binding protein 1 (PEBP1) - a suppressor of the BRAF/MEK/ERK pathway - via RIPK4 observed in pancreatic cancer did not occur in melanoma. Neither downregulation nor upregulation of RIPK4 in BRAF- mutated cells affected PEBP1 levels or the BRAF/MEK/ERK pathway. The downregulation of RIPK4 inhibited cell proliferation and the FAK/AKT pathway, and increased BRAFi efficiency in WM266.4 cells. However, the silencing of RIPK4 did not induce apoptosis or necroptosis. Our study suggests that RIPK4 may be an off-target for BRAF inhibitors

CKS1 expression in melanocytic nevi and melanoma

Cyclin-dependent kinase subunit 1 (Cks1) regulates the degradation of p27, an important G1-S inhibitor, which is up regulated by MAPK pathway activation. In this study, we sought to determine whether Cks1 expression is increased in melanocytic tumors and correlates with outcome and/or other clinicopathologic prognostic markers. Cks1 expression was assessed by immunohistochemistry in 298 melanocytic lesions. The frequency and intensity of cytoplasmic and nuclear expression was scored as a labeling index and correlated with clinico-pathological data. Nuclear Cks1 protein was found in 63% of melanocytic nevi, 89% primary and 90% metastatic melanomas with mean labeling index of 7 ± 16, 19 ± 20, and 30 ± 29, respectively. While cytoplasmic Cks1 was found in 41% of melanocytic nevi, 84% primary and 95% metastatic melanomas with mean labeling index of 18 ± 34, 35 ± 34, and 52 ± 34, accordingly. Histologic stepwise model of tumor progression, defined as progression from benign nevi to primary melanomas, to melanoma metastases, revealed a significant increase in nuclear and cytoplasmic Cks1 expression with tumor progression. Nuclear and cytoplasmic Cks1 expression correlated with the presence of ulceration, increased mitotic rate, Breslow depth, Clark level, tumor infiltrating lymphocytes and gender. However, other well-known prognostic factors (age, anatomic site, and regression) did not correlate with any type of Cks1 expression. Similarly, increasing nuclear expression of Cks1 significantly correlated with worse overall survival. Thus, Cks1 expression appears to play a role in the progression of melanoma, where high levels of expression are associated with poor outcome. Cytoplasmic expression of Cks1 might represent high turnover of protein via the ubiquination/proteosome pathway. © Brozyna et al

Melanogenesis is directly affected by metabolites of melatonin in human melanoma cells

Melatonin (N-acetyl-5-methoxytryptamine, MEL), its kynurenic ($N^{1}$-acetyl-$N^{2}$-formyl-5-methoxykynurenine, AFMK) and indolic derivatives (6-hydroxymelatonin, 6(OH)MEL and 5-methoxytryptamine, 5-MT) are endogenously produced in human epidermis. Melatonin, produced by the pineal gland, brain and peripheral organs, displays a diversity of physiological functions including anti-inflammatory, immunomodulatory, and anti-tumor capacities. Herein, we assessed their regulatory effect on melanogenesis using amelanotic (A375, Sk-Mel-28) and highly pigmented (MNT-1, melanotic) human melanoma cell lines. We discovered that subjected compounds decrease the downstream pathway of melanin synthesis by causing a significant drop of cyclic adenosine monophosphate (cAMP) level, the microphthalmia-associated transcription factor (MITF) and resultant collapse of tyrosinase (TYR) activity, and melanin content comparatively to N-phenylthiourea (PTU, a positive control). We observed a reduction in pigment in melanosomes visualized by the transmission electron microscopy. Finally, we assessed the role of G-protein-coupled seven-transmembrane-domain receptors. Obtained results revealed that nonselective MT1 and MT2 receptor antagonist (luzindole) or selective MT2 receptor antagonist (4-P-PDOT) did not affect dysregulation of the melanin pathway indicating a receptor-independent mechanism. Our findings, together with the current state of the art, provide a convenient experimental model to study the complex relationship between metabolites of melatonin and the control of pigmentation serving as a future and rationale strategy for targeted therapies of melanoma-affected patients

Vitamin D endocrine system in breast cancer

Vitamin D is a steroid hormone of great importance in the human body. It is produced in the skin from 7-dehydrocholesterol, upon UV radiation. In order to exert its functions, vitamin D has to be hydroxylated (via CYP27A1 and CYP27B1 hydroxylases), which is followed by its interaction with the vitamin D receptor (VDR) or retinoic acid-related orphan receptors a or gamma (ROR alpha and ROR gamma). By binding with the vitamin D response elements (VDRE) located in the promoter regions, the vitamin D ligand-receptor complex may regulate vitamin D-related genes. Recently, vitamin D has acquired a great interest for its plausible association with cancer development. This review discusses the potential role of vitamin D, its analogues, and enzymes involved in its metabolism with breast cancer incidence and outcome. According to the literature, alterations in the vitamin D endocrine system, both at the mRNA and protein level, have an impact on breast cancer incidence and prognosis. Moreover, specific enzymes participating in vitamin D metabolism may serve as therapeutic targets. Notably, treatment with vitamin D analogues also gives promising results in experimental research. However, given the fact that breast cancer is heterogenous disease, further studies are needed to thoroughly elucidate the potential of vitamin D and enzymes involved in its metabolism in breast cancer development, progression and therapy. Therefore, plausible effects of vitamin D in cancer therapy or prevention have been the principal aim of numerous studies

Computational Analysis Identifies Novel Biomarkers for High-Risk Bladder Cancer Patients

In the case of bladder cancer, carcinoma in situ (CIS) is known to have poor diagnosis. However, there are not enough studies that examine the biomarkers relevant to CIS development. Omics experiments generate data with tens of thousands of descriptive variables, e.g., gene expression levels. Often, many of these descriptive variables are identified as somehow relevant, resulting in hundreds or thousands of relevant variables for building models or for further data analysis. We analyze one such dataset describing patients with bladder cancer, mostly non-muscle-invasive (NMIBC), and propose a novel approach to feature selection. This approach returns high-quality features for prediction and yet allows interpretability as well as a certain level of insight into the analyzed data. As a result, we obtain a small set of seven of the most-useful biomarkers for diagnostics. They can also be used to build tests that avoid the costly and time-consuming existing methods. We summarize the current biological knowledge of the chosen biomarkers and contrast it with our findings