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)

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

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization

Publisher Copyright: Copyright © 2021 The Authors, some rights reserved;In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.Peer reviewe

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-1β 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-κB signaling in a RIPK4-dependent (RIPK4high) or independent (RIPK4low) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial)

Regulation of selected transcripts' levels by MCPIP1

Nowotwór nerki stanowi zaledwie 4% ogółu zachorowań na nowotwory złośliwe, z czego najczęstszym podtypem histopatologicznym jest rak jasnokomórkowy nerki. Jest to nowotwór o wysokim współczynniku śmiertelności. Ze względu na fakt, że tradycyjne metody leczenia często są nieskuteczne, istnieją próby rozwoju nowych i bardziej obiecujących terapii. Najnowsze dane wskazują na obniżony poziom białka MCPIP1 w przypadku ccRCC. MCPIP1 jest białkiem wykazującym aktywność RNazową, która wynika z obecności domeny PIN. Uważa się, że główną funkcją MCPIP1 jest negatywna kontrola stanu zapalnego, choć obserwuje się także jego udział w procesie angiogenezy czy różnicowania się komórek. Celem niniejszej pracy było poznanie czy nadekspresja MCPIP1 prowadzi do obniżenia poziomu mRNA kodujących białka uczestniczące w transdukcji sygnału tj. NGEF1, RIPK4, NDRG2, SGK2, GPRC5B oraz TSC22D3. Nadekspresja została wykonana poprzez wprowadzenie cDNA kodujące aktywne białko MCPIP1 oraz formę zmutowaną z mutacją punktową inaktywującą domenę PIN (D141N) do komórek Caki-1. Ekspresja regulowana była poprzez system TetON. Ocenę poziomu badanych transkryptów wykonano przy pomocy qRT-PCR. Następnie zanalizowano in silico region 3’UTR analizowanych mRNA w celu przewidzenia obecności potencjalnej struktury pętli rozpoznawanej przez MCPIP1. Uzyskane wyniki sugerują udział MCPIP1 w obniżeniu poziomu badanych transkryptów. W przypadku SGK2 oraz RIPK4 wydaje się, że MCPIP1 może bezpośrednio wpływać na poziom ich transkryptów w komórce. Z kolei w przypadku NGEF1, GPRC5B, NDRG2 oraz TSC22D3 prawdopodobnie MCPIP1 reguluje poziom mRNA kodujące białka odpowiedzialne za regulację poziomu analizowanych transkryptów, co wynika ze znacznego wzrostu ich poziomu w komórkach z nadekspresją MCPIP1 z inaktywowaną domeną PIN. W przypadku mRNA dla NDRG2 oraz TSC22D3 otrzymane wyniki nie były istotne statystycznie.Uzyskane wyniki pokazują, że MCPIP1 obniża poziom wybranych transkryptów w sposób bezpośredni poprzez mechanizm zależny od domeny PIN lub pośrednio poprzez modulację transkryptów kodujących białka regulatorowe, takie jak czynniki transkrypcyjne.Renal cell carcinoma accounts for merely 4% of all malignant cancers, which the most common histological subtype is clear cell renal cell carcinoma. This type of cancer features high mortality rate. Due to the fact that traditional therapy is often ineffective, there are trials of development of new, more promising therapies. Recent data show the reduction of MCPIP1 protein level in ccRCC. MCPIP1 is a protein with RNase activity, which results from presence of a PIN domain. It is believed that the main function of MCPIP1 is a negative control of inflammation, but on the other hand, MCPIP1 is also involved in angiogenesis and cell differentiation. The aim of this study was to investigate whether overexpression of MCPIP1 leads to reduction of mRNA level encoding proteins involved in signal transduction i.e. NGEF1, RIPK4, NDRG2, SGK2, GPRC5B and TSC22D3. Overexpression has been achieved by introducing cDNA encoding active MCPIP1 protein and its mutant form with a point mutation inactivating PIN domain (D141N) into Caki-1 cells. Expression was regulated by TetOn system. Assessment of examined transcripts level was measured using qRT-PCR. Moreover, in silico analysis was performed to predict the presence of loop structure in 3'UTRs for analyzed mRNA which can be a target for MCPIP1. The results suggest that MCPIP1 contributes to reduction of degree of examined transcripts. It appears, that MCPIP1 can directly affect level of SGK2 and RIPK4 transcripts within the cell. However, in case of NGEF, GPRCB, NDRG2 and TSC22D3, it seems, that MCPIP1 regulates the level of mRNA coding for proteins responsible for regulation of analyzed transcripts, what is manifested by a significant increase of their level in cells overexpressing MCPIP1 with inactive PIN domain. In case of NDRG2 and TSC22D3 mRNAs, obtained results were statistically insignificant.Obtained results show that MCPIP1 downregulates selected transcripts directly, by PIN-domain dependent mechanism or indirectly, by modulation of transcripts coding for regulatory proteins, such as transcription factors