Crk-like adapter protein is overexpressed in cervical carcinoma, facilitates proliferation, invasion and chemoresistance, and regulates Src and Akt signaling
Abstract:Overexpression of Crk-like (CrkL) adapter protein has been implicated in a number of types of human cancer. However, its involvement in human cervical carcinoma remains unclear. The present study aimed to explore the clinical significance and biological characteristics of CrkL in human cervical carcinoma. CrkL protein expression was examined in tissue samples from 92 cases of cervical carcinoma using immunohistochemistry, and was found to be overexpressed in 48.9% (45/92 cases). CrkL was transfected into HeLa … Show more
“…3b), consistent with a possible role of CRKL gene in facilitating the proliferation of tumor cells. Our hypothesis and results were consistent with previous report as well [68]. Fig.…”
Section: Resultssupporting
confidence: 94%
“…We further explored the potential function of CRKL in regulating alternative splicing in HeLa cells using shRNA to knock-down CRKL expression. The results confirmed the role of CRKL in promoting cell proliferation in HeLa cell published recently [68], and also showed that CRKL could regulate the alternative splicing of pre-mRNAs from hundreds of genes. We further showed that 94% of CRKL-regulated alternative splicing events detected in HeLa cells could be validated by RT-qPCR approach.…”
Section: Introductionsupporting
confidence: 90%
“…It has been reported recently that overexpressed CRKL promotes the phosphorylation of AKT [68, 84]. AKT is a serine-threonine (SR) protein kinase and regulates SR protein kinase activity [74, 85, 86].…”
Section: Resultsmentioning
confidence: 99%
“…CRKL is overexpressed in a number of types of human malignant tumors, including cervical cancer, lung cancer, breast cancer, gastric cancer, and pancreatic carcinoma. It plays crucial roles in tumorigenesis and cancer progression [65–68, 87]. Regulation of gene transcription and alternative splicing by key kinases and adaptors protein in signaling transduction pathways has been extensively studied [69–74].…”
Section: Discussionmentioning
confidence: 99%
“…CRKL is overexpressed in various types of human cancer and can induce cancer cell proliferation and invasion [64–67]. In addition, CRKL was demonstrated to be an oncoprotein contributing to malignant cell growth and chemoresistance and promoting cancer cell invasion through a Src-dependent pathway [68].…”
Background
Aberrant spliced isoforms are specifically associated with cancer progression and metastasis. The cytoplasmic adaptor
CRKL
(v-crk avian sarcoma virus CT10 oncogene homolog-like) is a CRK like proto-oncogene, which encodes a SH2 and SH3 (src homology) domain-containing adaptor protein. CRKL is tightly linked to leukemia via its binding partners BCR-ABL and TEL-ABL, upregulated in multiple types of human cancers, and induce cancer cell proliferation and invasion. However, it remains unclear whether signaling adaptors such as CRKL could regulate alternative splicing.
Methods
We analyzed the expression level of
CRKL
in 305 cervical cancer tissue samples available in TCGA database, and then selected two groups of cancer samples with CRKL differentially expressed to analyzed potential CRKL-regulated alternative splicing events (ASEs). CRKL was knocked down by shRNA to further study CRKL-regulated alternative splicing and the activity of SR protein kinases in HeLa cells using RNA-Seq and Western blot techniques. We validated 43 CRKL-regulated ASEs detected by RNA-seq in HeLa cells, using RT-qPCR analysis of HeLa cell samples and using RNA-seq data of the two group of clinical cervical samples.
Results
The expression of
CRKL
was mostly up-regulated in stage I cervical cancer samples. Knock-down of
CRKL
led to a reduced cell proliferation. CRKL-regulated alternative splicing of a large number of genes were enriched in cancer-related functional pathways, among which DNA repair and G2/M mitotic cell cycle, GnRH signaling were shared among the top 10 enriched GO terms and KEGG pathways by results from clinical samples and HeLa cell model. We showed that CRKL-regulated ASEs revealed by computational analysis using ABLas software in HeLa cell were highly validated by RT-qPCR, and also validated by cervical cancer clinical samples.
Conclusions
This is the first report of CRKL-regulation of the alternative splicing of a number of genes critical in tumorigenesis and cancer progression, which is consistent with CRKL reported role as a signaling adaptor and a kinase. Our results underline that the signaling adaptor CRKL might integrate the external and intrinsic cellular signals and coordinate the dynamic activation of cellular signaling pathways including alternative splicing regulation.
Electronic supplementary material
The online version of this article (10.1186/s12885-019-5671-8) contains supplementary material, which is available to authorized users.
“…3b), consistent with a possible role of CRKL gene in facilitating the proliferation of tumor cells. Our hypothesis and results were consistent with previous report as well [68]. Fig.…”
Section: Resultssupporting
confidence: 94%
“…We further explored the potential function of CRKL in regulating alternative splicing in HeLa cells using shRNA to knock-down CRKL expression. The results confirmed the role of CRKL in promoting cell proliferation in HeLa cell published recently [68], and also showed that CRKL could regulate the alternative splicing of pre-mRNAs from hundreds of genes. We further showed that 94% of CRKL-regulated alternative splicing events detected in HeLa cells could be validated by RT-qPCR approach.…”
Section: Introductionsupporting
confidence: 90%
“…It has been reported recently that overexpressed CRKL promotes the phosphorylation of AKT [68, 84]. AKT is a serine-threonine (SR) protein kinase and regulates SR protein kinase activity [74, 85, 86].…”
Section: Resultsmentioning
confidence: 99%
“…CRKL is overexpressed in a number of types of human malignant tumors, including cervical cancer, lung cancer, breast cancer, gastric cancer, and pancreatic carcinoma. It plays crucial roles in tumorigenesis and cancer progression [65–68, 87]. Regulation of gene transcription and alternative splicing by key kinases and adaptors protein in signaling transduction pathways has been extensively studied [69–74].…”
Section: Discussionmentioning
confidence: 99%
“…CRKL is overexpressed in various types of human cancer and can induce cancer cell proliferation and invasion [64–67]. In addition, CRKL was demonstrated to be an oncoprotein contributing to malignant cell growth and chemoresistance and promoting cancer cell invasion through a Src-dependent pathway [68].…”
Background
Aberrant spliced isoforms are specifically associated with cancer progression and metastasis. The cytoplasmic adaptor
CRKL
(v-crk avian sarcoma virus CT10 oncogene homolog-like) is a CRK like proto-oncogene, which encodes a SH2 and SH3 (src homology) domain-containing adaptor protein. CRKL is tightly linked to leukemia via its binding partners BCR-ABL and TEL-ABL, upregulated in multiple types of human cancers, and induce cancer cell proliferation and invasion. However, it remains unclear whether signaling adaptors such as CRKL could regulate alternative splicing.
Methods
We analyzed the expression level of
CRKL
in 305 cervical cancer tissue samples available in TCGA database, and then selected two groups of cancer samples with CRKL differentially expressed to analyzed potential CRKL-regulated alternative splicing events (ASEs). CRKL was knocked down by shRNA to further study CRKL-regulated alternative splicing and the activity of SR protein kinases in HeLa cells using RNA-Seq and Western blot techniques. We validated 43 CRKL-regulated ASEs detected by RNA-seq in HeLa cells, using RT-qPCR analysis of HeLa cell samples and using RNA-seq data of the two group of clinical cervical samples.
Results
The expression of
CRKL
was mostly up-regulated in stage I cervical cancer samples. Knock-down of
CRKL
led to a reduced cell proliferation. CRKL-regulated alternative splicing of a large number of genes were enriched in cancer-related functional pathways, among which DNA repair and G2/M mitotic cell cycle, GnRH signaling were shared among the top 10 enriched GO terms and KEGG pathways by results from clinical samples and HeLa cell model. We showed that CRKL-regulated ASEs revealed by computational analysis using ABLas software in HeLa cell were highly validated by RT-qPCR, and also validated by cervical cancer clinical samples.
Conclusions
This is the first report of CRKL-regulation of the alternative splicing of a number of genes critical in tumorigenesis and cancer progression, which is consistent with CRKL reported role as a signaling adaptor and a kinase. Our results underline that the signaling adaptor CRKL might integrate the external and intrinsic cellular signals and coordinate the dynamic activation of cellular signaling pathways including alternative splicing regulation.
Electronic supplementary material
The online version of this article (10.1186/s12885-019-5671-8) contains supplementary material, which is available to authorized users.
Background
Gastrointestinal (GI)‐neuroendocrine neoplasms (NENs) are subclassified in neuroendocrine tumors (NETs), neuroendocrine carcinomas (NECs), and mixed neuroendocrine–non‐neuroendocrine neoplasms (MiNENs). The genetic characteristics of GI‐NEN has been a hot issue in recent years, but more studies are needed to provide further details. This study aims to provide additional data about genomic characteristics of GI‐NENs and the genetic differences between NETs and NECs.
Patients and Methods
Thirteen samples were selected for next‐generation sequencing (NGS) analysis with a 425‐gene panel. Microsatellite instability (MSI) and tumor mutational burden (TMB) were calculated as well as immunohistochemistry (IHC) was used to test for protein expression.
Results
Genetic alterations were very common in NECs, but rare in NETs. The average TMB of NETs and NECs was 2.3 and 6.9, respectively. The TMB of NECs was significantly higher compared to NETs. The TP53 mutation rate was significantly higher in NECs than in NETs (100% vs. 20%), other mutations involved MTOR (n = 2, 15.4%), DDR2 (n = 3, 23.1%), ERBB4 (n = 1, 7.7%), BRCA1 (n = 1, 7.7%), BRCA2 (n = 1, 7.7%), ATM (n = 1, 7.7%), and SMAD4 (n = 1, 7.7%). Deep loss of SMAD4 (1/3, 33.3%), SDHB (1/3, 33.3%), RB1 (1/3, 33.3%), and BRCA2 (1/3, 33.3%), high‐level amplification of CRKL (1/3, 33.3%), CCNE1(1/3, 33.3%), and MCL1(1/3, 33.3%) were found in NECs. The integrated analysis found these genetic alterations frequently involve DNA repair and cell cycle, PI3K/AKT/mTOR and TGF‐β/SMAD4 signaling pathways.
Conclusion
Genetic alterations were very common in NECs and rare in NETs, and frequently involved three main signaling pathways. NEC patients harboring these genetic alterations may benefit from targeted therapy and PD‐1/PD‐L1 immunotherapy.
The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors. Here, we knocked down Crk, CrkL, or both using siRNAs in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes. The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells. These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.
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