KIN17 functions in DNA damage repair and chemosensitivity by modulating RAD51 in hepatocellular carcinoma.

The limited response of hepatocellular carcinoma (HCC) to chemotherapy drugs has always been a bottleneck in therapy. DNA damage repair is a major reason for chemoresistance. Previous studies have confirmed that KIN17 affects chemosensitivity. In this study, we examined the impact of KIN17 on chemotherapy response and DNA repair in HCC cells treated with oxaliplatin (L-OHP). We evaluated the expression and biological roles of KIN17 in HCC using bioinformatic analysis. The correlation between KIN17 and RAD51, particularly their nuclear expression levels, was evaluated using immunofluorescence, immunoblotting after nucleocytoplasmic separation in HCC cells, and immunohistochemistry of mouse xenograft tumors and human HCC tissues. The results indicated a significant increase in KIN17 expression in HCC tissues compared to normal tissues. The GSEA analysis revealed that upregulation of KIN17 was significantly associated with DNA damage repair. Knockdown of KIN17 led to increased DNA damage and reduced cellular survival after exposure to L-OHP. On the other hand, overexpression of KIN17 was linked to decreased DNA damage and improved cell survival following L-OHP treatment. Further experiments indicated that KIN17 affects the expression of RAD51, particularly in the nucleus. KIN17 plays a crucial role in influencing the sensitivity of HCC to chemotherapy by triggering the DNA repair response. Increased expression of KIN17 is associated with a poor prognosis for HCC patients, indicating that KIN17 could serve as a prognostic marker and therapeutic target for HCC.

Kin17 regulates proper cortical localization of Miranda in Drosophila neuroblasts by regulating Flfl expression.

During asymmetric division of Drosophila larval neuroblasts, the fate determinant Prospero (Pros) and its adaptor Miranda (Mira) are segregated to the basal cortex through atypical protein kinase C (aPKC) phosphorylation of Mira and displacement from the apical cortex, but Mira localization after aPKC phosphorylation is not well understood. We identify Kin17, a DNA replication and repair protein, as a regulator of Mira localization during asymmetric cell division. Loss of Kin17 leads to aberrant localization of Mira and Pros to the centrosome, cytoplasm, and nucleus. We provide evidence to show that the mislocalization of Mira and Pros is likely due to reduced expression of Falafel (Flfl), a component of protein phosphatase 4 (PP4), and defects in dephosphorylation of serine-96 of Mira. Our work reveals that Mira is likely dephosphorylated by PP4 at the centrosome to ensure proper basal localization of Mira after aPKC phosphorylation and that Kin17 regulates PP4 activity by regulating Flfl expression.

Roles and regulatory mechanisms of KIN17 in cancers (Review).

KIN17, which is known as a DNA and RNA binding protein, is highly expressed in numerous types of human cancers and was discovered to participate in several vital cell behaviors, including DNA replication, damage repair, regulation of cell cycle and RNA processing. Furthermore, KIN17 is associated with cancer cell proliferation, migration, invasion and cell cycle regulation by regulating pathways including the p38 MAPK, NF-κB-Snail and TGF-ß/Smad2 signaling pathways. In addition, knockdown of KIN17 was found to enhance the sensitivity of tumor cells to chemotherapeutic agents. Immunohistochemical analysis revealed that there were significant differences in the expression of KIN17 between cancer tissues and adjacent tissues. Both the Kaplan-Meier survival analysis and multivariate Cox regression analysis indicated that KIN17 is aberrantly high expressed in various tumor tissues and is also associated with poor prognosis in patients with various tumor types. Taken together, KIN17 has key roles in tumorigenesis and cancer development. Investigating the relationship between KIN17 and neoplasms will provide a vital theoretical basis for KIN17 to serve as a diagnostic and prognostic biomarker for cancer patients and as a potential target for cancer therapy.

KIN17 promotes cell migration and invasion through stimulating the TGF-ß/Smad2 pathway in hepatocellular carcinoma.

KIN17 DNA and RNA binding protein (Kin17) is involved in the regulation of tumorigenesis of diverse human cancers. However, its role in the cancer progression and metastasis in hepatocellular carcinoma (HCC) remains largely unknown. Bioinformatics and immunohistochemistry staining were used to investigate the expression pattern of KIN17 and its prognostic value in HCC patients. The transwell, wound-healing assay was employed to determine the effects of KIN17 on migration and invasion of HCC cells in vitro. The tail veins model was employed to determine the effects of KIN17 on lung metastasis in vivo. The biological mechanisms involved in cell migration and invasion regulated by KIN17 were determined with Western blot analysis method. KIN17 expression was significantly increased in HCC tissues compared with adjacent normal tissues, with particularly higher in portal vein tumor thrombus and intrahepatic metastasis tissues. Patients with higher KIN17 expression experienced poor overall and disease free survival. KIN17 knockdown in HuH7 and HepG2 cells significantly reduced cell migration and invasion abilities, whereas its overexpression promoted migration and invasion in MHCC-97L and HepG2 cells in vitro and in vivo. In HuH7 and HepG2 cells, KIN17 knockdown inhibited the TGF-ß/Smad2 pathway. In contrast, KIN17 overexpression stimulated TGF-ß/Smad2 pathway in MHCC-97L and HepG2 cells, along with the genes involved in the epithelial-mesenchymal transition. These findings suggest that KIN17 promotes migration and invasion in HCC cells by stimulating the TGF-ß/Smad2 pathway. KIN17 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.

Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein.

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein-protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.

KIN17 promotes tumor metastasis by activating EMT signaling in luminal-A breast cancer.

BACKGROUND: Breast cancer (BC), the most common cause of cancer death in women, overtook lung cancer as the leading cause of cancer worldwide in 2020. Although many studies have proposed KIN17 as a biomarker of tumorigenesis in different cancer types, its role in tumor metastasis, particularly in BC metastasis, has been underexplored. This study aimed to explore the role of KIN17 in BC metastasis. METHODS: Survival analyses was performed to identify the association between KIN17 expression and BC patient survival in silico. Using lentivirus constructs, we developed bidirectional KIN17 expression (KD, knockdown; OE, overexpression) cellular models of luminal-A (Lum-A) breast cancer MCF-7 cells. We performed in vitro wound healing, transwell with and without Matrigel assays, and in vivo tail-vein metastasis assay to evaluate the migration and invasion abilities of MCF-7 with stable KIN17 knockdown or overexpression. Western blotting was performed to compare the changes in protein expression. RESULTS: We found that KIN17 expression was associated with poor overall survival (OS), relapse-free survival (RFS), distant metastasis-free survival (DMFS) and post-progression survival (PPS), particularly in Lum-A breast cancer patients. Later, we found that KIN17 knockdown inhibited migration and invasion of MCF-7 cells via regulating EMT-associated signaling pathways in vitro and decreases metastatic spread of the disease in vivo. In contrast, KIN17 overexpression promoted migration and invasion of MCF-7 cells in vitro and increased the metastatic spread of the disease in vivo. CONCLUSIONS: Overall, our findings provide preliminary data which suggests KIN17 of importance to target in metastatic Lum-A patients.

Analysis of the association between KIN17 expression and the clinical features/prognosis of epithelial ovarian cancer, and the effects of KIN17 in SKOV3 cells.

DNA double-strand breaks (DSBs) are an important mechanism of chemotherapy in epithelial ovarian cancer (EOC). Kin17 DNA and RNA binding protein (KIN17) serves a crucial role in DSB repair. In the present study, the association between KIN17 and EOC, and the effects of KIN17 on EOC cells in vitro were evaluated. A bioinformatics method was used to determine the mRNA expression levels of KIN17 in EOC and its association with EOC prognosis including overall survival (OS) and progression free survival (PFS) time. Western blotting and immunohistochemical staining were used to evaluate the expression levels of KIN17 in EOC samples. Kaplan-Meier and Cox regression analyses were utilized to analyze risk factors for the OS of patients with EOC. A Cell Counting Kit-8 assay was performed to explore the roles of KIN17 in SKOV3 cells. Both the transcription and expression of KIN17 were upregulated in EOC tissues. Furthermore, the OS of patients with EOC with high mRNA expression levels of KIN17 was shorter than that of patients with EOC with low expression levels. High KIN17 expression was an independent risk factor for EOC prognosis. Furthermore, KIN17 knockdown inhibited the proliferation of SKOV3 cells, enhanced the sensitivity of the cells to cisplatin and inhibited the migration ability of the cells. These results suggested that KIN17 may act as an ideal candidate for therapy and as a prognostic biomarker of EOC, although the underlying mechanisms require further exploration.

Kin17 facilitates thyroid cancer cell proliferation, migration, and invasion by activating p38 MAPK signaling pathway.

Kin17 DNA and RNA binding protein (Kin17) is an extremely conserved nuclear protein that is almost expressed in every type of mammal cells. Recently, Kin17 has been implicated into the regulation of tumorigenesis of diverse human cancers. However, its functions in thyroid cancer (TC) are still largely unexplored. Kin17 mRNA and protein level were tested by qRT-PCR and western blot, respectively. Effects of Kin17 on TC cell proliferation were estimated by colony formation assay and flow cytometry analysis in vitro as well as by in vivo tumor growth experiment. TC cell migratory and invasive capacities were assessed via wound-healing and transwell experiments. Epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin and N-cadherin) and p38 MAPAK signaling pathway-related proteins (p-p38, p38, Cyclin D1, and p27) were examined via western blot. Kin17 was remarkably increased in TC tissue samples and cell lines at both mRNA and protein levels compared to normal tissue and control cell line. Knockdown of Kin17 obviously repressed TC cell proliferation, arrested cell cycle, and inhibited TC cell migration and invasion in vitro, while overexpression of Kin17 produced opposite effects. Kin17 knockdown suppressed p38 MAPK signaling pathway, while Kin17 overexpression activated this pathway. Treatment of p38 agonist (p79350) abolished the repressive effects of sh-Kin17 on TC cell proliferation, migration, and invasion, as well as on p38 pathway. Kin17 knockdown was also found to enhance the sensitivity of Doxorubicin of TC cells. In addition, Kin17 knockdown in vivo also markedly repressed TC tumor growth and p38 pathway. Kin17 functioned as an oncogene of TC by activating p38 MAPK signaling pathway.

Kin17 knockdown suppresses the migration and invasion of cervical cancer cells through NF-κB-Snail pathway.

Cervical cancer is one of the most common cancers in women worldwide. Metastasis in cancer has been a Gordian knot due to unsatisfactory clinical treatments. KIN17, a highly conserved gene from yeast to human, up-regulation is associated with the pathogenesis and development of several common cancers. Our previous works revealed that elevated expression of kin17 observed in cervical cancer tissues showed a close association with lymph node metastasis. This study aimed to explore roles and mechanisms of kin17 in the migration and invasion of cervical cancer cells. Cervical cancer cell lines HeLa and SiHa with kin17 knockdown were constructed by using recombinant lentiviral vector that carry specific siRNA targeting KIN17 gene. The mRNA and protein levels of kin17 in cells were determined by RT-qPCR and western blotting, respectively. Wound healing assay and transwell assays were performed to assess the migration and invasion abilities of the cancer cells, respectively. The expression of signaling proteins involved in the NF-κB-Snail pathway was analyzed by western blotting. As our results showed, the mRNA and protein levels of kin17 in HeLa cells and SiHa cells showed a significant decrease by transfection with recombinant lentiviral vector carrying specific siRNA. Compared with control group, the migration rates were decreased in the kin17 knockdown group in both HeLa and SiHa cell lines in wound healing assay as well as transwell assay without matrigel. Kin17 knockdown also reduced the cell invasion number of both HeLa and SiHa cells. In addition, the phosphorylation of nuclear factor Kαppa B (NF-κB) p65, IKαppa B kinase α (IKKα), and IKαppa B α (IκBα) in NF-κB pathway and the expression of Snail were decreased in HeLa cells and SiHa cells by kin17 knockdown. Our results demonstrated that knockdown of kin17 in cervical cancer cells suppressed cell migration and invasion, and inhibited the activity of NF-κB signaling pathway and the expression of Snail. These findings suggested kin17 as an essential regulator of the cell migration and invasion and the underlying molecular mechanism involved NF-κB-Snail pathway in cervical cancer. This might serve as a novel molecular therapeutic target for treating cervical cancer metastasis.

Biophysical characterization and molecular phylogeny of human KIN protein.

The DNA/RNA-binding KIN protein was discovered in 1989, and since then, it has been found to participate in several processes, e.g., as a transcription factor in bacteria, yeasts, and plants, in immunoglobulin isotype switching, and in the repair and resolution of double-strand breaks caused by ionizing radiation. However, the complete three-dimensional structure and biophysical properties of KIN remain important information for clarifying its function and to help elucidate mechanisms associated with it not yet completely understood. The present study provides data on phylogenetic analyses of the different domains, as well as a biophysical characterization of the human KIN protein (HSAKIN) using bioinformatics techniques, circular dichroism spectroscopy, and differential scanning calorimetry to estimate the composition of secondary structure elements; further studies were performed to determine the biophysical parameters ΔHm and Tm. The phylogenetic analysis indicated that the zinc-finger and winged helix domains are highly conserved in KIN, with mean identity of 90.37% and 65.36%, respectively. The KOW motif was conserved only among the higher eukaryotes, indicating that this motif emerged later on the evolutionary timescale. HSAKIN has more than 50% of its secondary structure composed by random coil and ß-turns. The highest values of ΔHm and Tm were found at pH 7.4 suggesting a stable structure at physiological conditions. The characteristics found for HSAKIN are primarily due to its relatively low composition of α-helices and ß-strands, making up less than half of the protein structure.

Knockdown of DNA/RNA-binding protein KIN17 promotes apoptosis of triple-negative breast cancer cells.

Effective therapy for breast cancer has been extensively studied worldwide, particularly for triple-negative breast cancer and drug-resistant subtypes. DNA/RNA-binding protein KIN17 (kin17) has been reported to be significantly upregulated in breast cancer cells, and is proposed to serve a role in the regulation of cell proliferation. The present study further investigated the association of kin17-knockdown with breast cancer cell apoptosis. Cell Counting kit-8, flow cytometry, TUNEL assay and caspase 3/7 analysis were performed on MDA-MB-231 cells to determine the association between kin17 and breast cancer cell apoptosis. In addition, western blot analysis was performed to investigate the mechanism of kin17 in the apoptosis of MDA-MB-231 cells. The results revealed that knockdown of kin17 inhibited proliferation and promoted apoptosis of MDA-MB-231 cells, and suggested a poly (adenosine diphosphate-ribose) polymerase-related mechanism behind the apoptosis of the cells. These findings suggested that kin17 could become a novel target for breast cancer therapy.

Upregulation of KIN17 is associated with non-small cell lung cancer invasiveness.

Kin17 DNA and RNA binding protein (Kin17) is a highly conserved protein that participates in DNA replication, DNA repair and cell cycle progression. Recently, the tumor-promoting function of Kin17 has been demonstrated and increasingly studied. In the present study, the role of Kin17 in the invasion and metastasis of non-small cells lung cancer (NSCLC) was investigated. Elevated Kin17 mRNA and protein expression was identified in a total of 97 NSCLC and benign lung lesion tissue specimens. Kin17 overexpression was significantly correlated with high tumor grade and lymph node metastasis, indicating poor patient prognosis. Scratch and Transwell assays demonstrated that the knockdown of KIN17 inhibited the ability of NSCLC cells to migrate and invade. Furthermore, reverse transcription-quantitative polymerase chain reaction and western blot analyses confirmed that knockdown of KIN17 decreased the expression of matrix metalloproteinase 7, epidermal growth factor receptor and v-myc avian myelocytomatosis viral oncogene homolog. The results of the present study indicate that Kin17 is markedly overexpressed in NSCLC tissues compared with benign lung lesion and peritumoral tissue. The upregulation of KIN17 may serve an important role in the metastasis of NSCLC cells. These results indicate that Kin17 is a novel diagnostic and prognostic biomarker of NSCLC, in addition to being a potential therapeutic target for the treatment of patients with NSCLC.

The kin17 Protein in Murine Melanoma Cells.

kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.

Expression of Kin17 promotes the proliferation of hepatocellular carcinoma cells in vitro and in vivo.

Kin17 protein is ubiquitously expressed in mammals and is correlated with vital biological functions. However, little is known about the role of Kin17 in the proliferation of hepatocellular carcinoma cells. The aim of the present study was to investigate whether the upregulation of Kin17 can promote the growth of hepatocellular carcinoma cells. A series of assays was performed to study the effect of Kin17 in the proliferation of hepatocellular carcinoma cells in vitro and in vivo. The western blotting results revealed that Kin17 expression was increased in hepatocellular carcinoma tissues compared with that of the corresponding normal tissues. Moreover, ectopic upregulation of Kin17 expression promoted the growth of hepatocellular carcinoma cells in vitro and in vivo. These results indicated that Kin17 is involved in the tumorigenesis of hepatocellular carcinoma, and that Kin17 has the potential to serve as a therapeutic target for hepatocellular carcinoma.

The Arabidopsis KIN17 and its homolog KLP mediate different aspects of plant growth and development.

Proteins harboring the kin17 domain (KIN17) constitute a family of well-conserved eukaryotic nuclear proteins involved in nucleic acid metabolism. In mammals, KIN17 orthologs contribute to DNA replication, RNA splicing, and DNA integrity maintenance. Recently, we reported a functional characterization of an Arabidopsis thaliana KIN17 homolog (AtKIN17) that uncovered a role for this protein in tuning physiological responses during copper (Cu) deficiency and oxidative stress. However, functions similar to those described in mammals may also be expected in plants given the conservation of functional domains in KIN17 orthologs. Here, we provide additional data consistent with the participation of AtKIN17 in controlling general plant growth and development, as well as in response to UV radiation. Furthermore, the Arabidopsis genome codes for a second homolog to KIN17, we referred to as KIN17-like-protein (KLP). KLP loss-of-function lines exhibited a reduced inhibition of root growth in response to copper excess and relatively elongated hypocotyls in etiolated seedlings. Altogether, our experimental data point to a general function of the kin17 domain proteins in plant growth and development.

Methylation of the DNA/RNA-binding protein Kin17 by METTL22 affects its association with chromatin.

Kin17 is a protein that was discovered through its immunoreactivity towards an antibody directed against prokaryotic RecA. Further study of Kin17 revealed a function in DNA replication and repair, as well as in pre-mRNA processing. Recently, it was found that Kin17 is methylated on lysine 135 by the newly discovered methyltransferase METTL22. To better understand the function of Kin17 and its regulation by methylation, we used multiple cell compartment protein affinity purification coupled with mass spectrometry (MCC-AP-MS) to identify novel interaction partners of Kin17 and to assess whether these interactions can take place on chromatin. Our results confirm that Kin17 interacts with METTL22 both in the soluble and chromatin fractions. We also show that many RNA-binding proteins, including the previously identified interactor BUD13 as well as spliceosomal and ribosomal subunits, associate with Kin17 in the soluble fraction. Interestingly, overexpression of METTL22 in HEK 293 cells displaces Kin17 from the chromatin to the cytoplasmic fraction, suggesting a role for methylation of lysine 135, a residue that lies within a winged helix domain of Kin17, in regulating association with chromatin. These results are discussed in view of the putative cellular function of Kin17. BIOLOGICAL SIGNIFICANCE: The results shown here broaden our understanding of METTL22, a member of a family of newly-discovered non-histone lysine methyltransferases and its substrate, Kin17, a DNA/RNA-binding protein with reported roles in DNA repair and replication and mRNA processing. An innovative method to study protein-protein interactions in multiple cell compartments is employed to outline the interaction network of both proteins. Functional experiments uncover a correlative role between Kin17 lysine methylation and its association with chromatin. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?