Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals <scp>CAPN</scp>2 centered network

Shen, Chengpin; Yu, Yanyan; Li, Hong; Yan, Guoquan; Liu, Mingqi; Shen, Huali; Yang, Pengyuan

doi:10.1002/pmic.201200027

Cited by 25 publications

(22 citation statements)

References 37 publications

(41 reference statements)

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“…This approach has been recently applied to analyze several proteolytic networks in humans (Niessen et al, 2011;Dix et al, 2012;Shen et al, 2012). Among others, PROTOMAP has also been used to study KLK4 substrates in a prostate cancer cell model, which identified KLK4 as a major regulator of several of TGFβ1-related proteins (Clements, 2013).…”

Section: Protein Topography and Migration Analysis Platformmentioning

confidence: 98%

Putative kallikrein substrates and their (patho)biological functions

Yu¹,

Prassas

Diamandis³

2014

Biological Chemistry

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Human tissue kallikreins (KLKs) represent the largest contiguous group of protease genes within our genome. All 15 KLK genes co-localize within approximately 260 kb in human chromosome 19q13.3-13.4 (14 640 kb→274 990 kb). They are widely expressed in several tissues and mediate a wide range of critical physiological and pathological processes. Despite the recent developments in KLK research, elucidation of their physiological substrate repertoires remains a largely unfulfilled goal. Phage display, positional scanning and combinatorial peptide library screens have provided some valuable insights into the preferred specificities of these powerful enzymes. More recently, advances in proteomic technologies have enabled more systemic approaches towards identification of KLK substrates in a physiological setting. The advent of degradomic technologies has brought to light several putative physiological substrates and has allowed a deeper appreciation of the in vivo functional roles of KLKs. The aim of this review is to provide an overview of the different techniques that have been utilized towards the elucidation of the substrate specificities of these enzymes and elaborate on their emerging in vivo substrates.

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Section: Protein Topography and Migration Analysis Platformmentioning

confidence: 98%

Putative kallikrein substrates and their (patho)biological functions

Yu¹,

Prassas

Diamandis³

2014

Biological Chemistry

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“…Three stepwise human metastatic HCC cell lines with a similar genetic backgrounds, including two subclones with high (MHCC97H) and low (MHCC97L) metastatic potential from MHCC97 [23, 24] and one even higher metastatic potential cell line (HCCLM3) from MHCC97H, were established using an in vivo clonal selection procedure [25]. The pulmonary metastatic rate was 100% in MHCC97H and HCCLM3 vs. 40% in MHCC97L [26].…”

Section: Resultsmentioning

confidence: 99%

“…An initial experiment revealed that none of the metastatic cell lines suffered from significantly reduced viability after 24 h of culturing in the serum-free medium [31]. Thus, the culture time of 24 h in DMEM CM without serum was identified as the essential conditions for culturing the cells [23]. The CM was centrifuged and precipitated with TCA.…”

Section: Resultsmentioning

confidence: 99%

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In-depth analysis of secretome and N-glycosecretome of human hepatocellular carcinoma metastatic cell lines shed light on metastasis correlated proteins

Jiang

Zhao

et al. 2016

Oncotarget

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Cancer cell metastasis is a major cause of cancer fatality. But the underlying molecular mechanisms remain incompletely understood, which results in the lack of efficient diagnosis, therapy and prevention approaches. Here, we report a systematic study on the secretory proteins (secretome) and secretory N-glycoproteins (N-glycosecretome) of four human hepatocellular carcinoma (HCC) cell lines with different metastatic potential, to explore the molecular mechanism of metastasis and supply the clues for effective measurement of diagnosis and therapy. Totally, 6242 unique gene products (GPs) and 1637 unique N-glycosites from 635 GPs were confidently identified. About 4000 GPs on average were quantified in each of the cell lines, 1156 of which show differential expression (p<0.05). Ninety-nine percentage of the significantly altered proteins were secretory proteins and proteins correlated to cell movement were significantly activated with the increasing of metastatic potential of the cell lines. Twenty-three GPs increased both in the secretome and the N-glycosecretome were chosen as candidates and verified by western blot analysis, and 10 of them were chosen for immunohistochemistry (IHC) analysis. The cumulative survival rates of the patients with candidate (FAT1, DKK3) suggested that these proteins might be used as biomarkers for HCC diagnosis. In addition, a comparative analysis with the published core human plasma database (1754 GPs) revealed that there were 182 proteins not presented in the human plasma database but identified by our studies, some of which were selected and verified successfully by western blotting in human plasma.

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“…Similarly, calpainmediated cleavage of FAK enhances cell adhesion and cell motility (30). A recent study identified enhanced proteolysis of ezrin, vimentin, and fibronectin in a highly metastatic cell line compared to that in a nonmetastatic counterpart (37). Some of these proteins can be specifically cleaved by calpain, including ezrin (38) and vimentin (39).…”

Section: Discussionmentioning

confidence: 99%

Calpain Genetic Disruption and HSP90 Inhibition Combine To Attenuate Mammary Tumorigenesis

Grieve

Gao

Hall

et al. 2016

Molecular and Cellular Biology

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Calpain is an intracellular Ca 2؉ -regulated protease system whose substrates include proteins involved in proliferation, survival, migration, invasion, and sensitivity to therapeutic drugs. Genetic disruption of calpain attenuated the tumorigenic potential of breast cancer cells and hypersensitized cells to 17AAG, an inhibitor of the molecular chaperone HSP90. Calpain-1 or -2 overexpression rendered cells resistant to 17AAG, whereas downregulation or inhibition of calpain-1/2 led to increased cell death in multiple breast cancer cell lines, including models of HER2 ؉ (SKBR3) and triple-negative basal-cell-like (MDA-MB-231) breast cancer. In an MDA-MB-231 orthotopic xenograft model, calpain knockdown or 17AAG treatment independently attenuated tumor growth and metastasis, while the combination was most effective. Calpain knockdown was associated with increased 17AAG-induced degradation of the HSP90 clients cyclin D1 and AKT and multidrug resistance protein 2, which correlated with increased expression of antimitogenic p27 KIP1 and proapoptotic BIM proteins. Like other therapeutics, 17AAG can be effluxed by specific ABC transporters. Calpain expression positively correlated with the expression of P glycoprotein in mouse embryonic fibroblasts. Importantly, we show that calpain affects ABC transporter function and efflux of clinically relevant doxorubicin. These observations provide a compelling rationale for exploring the combination of calpain inhibition with new or existing cancer therapeutics. Calpains are a family of intracellular calcium-dependent proteases. Of the 15 mammalian isoforms, calpain-1 and calpain-2 are ubiquitously expressed and have been most extensively studied (reviewed in reference 1). They are heterodimers consisting of isoform-specific catalytic subunits encoded by CAPN1 and CAPN2, respectively, and a common regulatory subunit encoded by CAPNS1. Heterodimerization is essential for both stability and enzymatic activity; hence, genetic disruption of CAPNS1 results in loss of both calpain-1 and -2 activities (2, 3).Elevated calpain expression or activity has been associated with multiple cancer types, including colon, liver, lung, prostate, ovarian, and breast cancers (reviewed in reference 4). Mechanistic understanding of calpain's involvement in cancer is confounded by its broad range of substrates, including signaling or structural proteins that regulate diverse cellular functions such as mitogenesis, migration, invasion, and death (1). Biomarker studies suggest a positive correlation between elevated calpain expression and a poor clinical outcome in breast cancer (5-7).Paradoxically, calpain is associated with prodeath (8, 9) or prosurvival (9-11), depending upon the physiologic context and specific challenges cells are exposed to. For example, calpain-mediated cleavage of PP2A supports activation of AKT and a prosurvival function in mouse embryonic fibroblasts (MEFs) (10), and in breast cancer cells and xenograft mouse models, knockdown of CAPN2 was associated with attenuated tumor growth...

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Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals CAPN2 centered network

Cited by 25 publications

References 37 publications

Putative kallikrein substrates and their (patho)biological functions

Putative kallikrein substrates and their (patho)biological functions

In-depth analysis of secretome and N-glycosecretome of human hepatocellular carcinoma metastatic cell lines shed light on metastasis correlated proteins

Calpain Genetic Disruption and HSP90 Inhibition Combine To Attenuate Mammary Tumorigenesis

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