Up-regulation of type I collagen during tumorigenesis of colorectal cancer revealed by quantitative proteomic analysis

717 Background: Increased collagen deposition and remodeling of the extracellular matrix has been shown to play a role in the pathology of gastrointestinal cancer (GC). The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) binds collagens and hereby regulates collagen fibrillogenesis. Matrix metalloproteinase (MMP) mediated cleavage of SPARC, increases the affinity for collagens up to 20-fold. SPARC has been shown to be overexpressed in GC patients and associated with GC cell invasion and metastasis. Increased expression and cleavage of SPARC might therefore be implicated in GC pathology by increasing collagen deposition. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific MMP-13 generated fragment of SPARC - a cleavage site involved in increased collagen affinity. The biomarker potential of this fragment was examined in serum from colorectal cancer (CRC) patients and healthy controls. Moreover, we evaluated the ability of cleaved SPARC to prevent type I collagen degradation in vitro. Methods: A monoclonal antibody was raised against a MMP-13-generated neo-epitope of SPARC and a competitive ELISA assay (SPARC-M) was developed and technically validated. Serum levels were assessed in CRC patients (n=50) and healthy controls (n=30). The ability of cleaved SPARC to prevent collagen degradation was investigated using an ELISA assay measuring type I collagen degradation by MMP-9. Results: SPARC-M was technically robust and specific for SPARC cleaved by MMP-13. The fragment was elevated in CRC patients when compared to healthy controls (p=0.0097). When MMP-13 degraded SPARC was incubated with type I collagen and MMP-9, type I collagen degradation was completely inhibited suggesting that SPARC increases collagen deposition by preventing collagen degradation. Conclusions: SPARC-M was significantly elevated in CRC patients compared to healthy controls suggesting biomarker potential. Biologically, cleaved SPARC may prevent type I collagen degradation hereby leading to a pro-tumorigenic environment. Larger clinical studies are needed to validate the clinical use of this biomarker in GC.

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Investigation of MSI status in acquired resistance to 5-fluorouracil treatment in colorectal cancer using a SILAC-based quantitative proteomic analysis method

Annals of Oncology ◽

10.1093/annonc/mdx393.061 ◽

2017 ◽

Vol 28 ◽

pp. v181

Author(s):

M. Ortega Duran ◽

S. Shnyder ◽

C. Sutton ◽

S-U. Shaheed

Keyword(s):

Colorectal Cancer ◽

Proteomic Analysis ◽

Acquired Resistance ◽

Analysis Method ◽

Quantitative Proteomic Analysis

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Proteomic Analysis Reveals a New Benefit of Periodic Mechanical Stress on Chondrocytes

Cellular Physiology and Biochemistry ◽

10.1159/000485652 ◽

2017 ◽

Vol 44 (4) ◽

pp. 1578-1590 ◽

Cited By ~ 1

Author(s):

Zeng Li ◽

Zhen Wang ◽

Shun Xu ◽

Wenwei Liang ◽

Weimin Fan

Keyword(s):

Mechanical Stress ◽

Proteomic Analysis ◽

Type I Collagen ◽

Glycogen Synthase ◽

Therapeutic Benefit ◽

Immunofluorescence Assay ◽

Differentially Expressed ◽

Type I ◽

Beta Catenin ◽

Underlying Mechanisms

Background/Aims: In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress. In our previous studies, we found that periodic mechanical stress can promote proliferation and matrix synthesis through the integrin beta 1-mediated ERK1/2 pathway, and we used proteomic analysis to detect quantitative changes in chondrocytes under periodic mechanical stress. Despite these results, the effects and mechanisms of periodic mechanical stress are still not fully understood, so in this study we extended our study using phosphoproteomic techniques. Methods: We used phosphoproteomic techniques to detect phosphorylation changes in chondrocytes under periodic mechanical stress and combined the results with the quantitative proteomic data to further explore the underlying mechanisms. Data were obtained by phosphorylation inhibition, quantitative real-time PCR (qPCR) analysis, western blot analysis and immunofluorescence assay. Results: From phosphoproteomic analysis, a total of 1073 phosphorylated proteins and 2054 phosphopeptides were identified. The number of significant differentially expressed proteins and phosphopeptides was 97 and 108, respectively (ratio >1.20 or <0.83 at p<0.05). Periodic mechanical stress increased glycogen synthase kinase 3-beta (GSK3-beta) phosphorylation at Y216, promoted the phosphorylation of beta-catenin, decreased beta-catenin levels and suppressed the expression of type I collagen. In contrast, inhibition of GSK3-beta by TWS119, which specifically inhibits the phosphorylation of Y216, suppressed the phosphorylation of beta-catenin, which resulted in the accumulation of beta-catenin and an increase in the expression of type I collagen. Conclusions: We successfully constructed differentially expressed phosphoproteomic profiles of rat chondrocytes under periodic mechanical stress, and discovered a potential new therapeutic benefit in which periodic mechanical stress suppressed the formation of type I collagen in the matrix of chondrocytes via phosphorylation of GSK3-beta and beta-catenin.

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Quantitative Proteomic Analysis of 2D and 3D Cultured Colorectal Cancer Cells: Profiling of Tankyrase Inhibitor XAV939-Induced Proteome

Scientific Reports ◽

10.1038/s41598-018-31564-6 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 7

Author(s):

Young Eun Kim ◽

Hyo Jin Jeon ◽

Dahee Kim ◽

Sun Young Lee ◽

Ki Young Kim ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Cells ◽

Proteomic Analysis ◽

Quantitative Proteomic Analysis ◽

Colorectal Cancer Cells ◽

2D And 3D

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Proteomic Investigation of Dermal Fibroblasts Isolated from Affected and Unaffected Skin Samples from Patients with Limited Cutaneous Systemic Sclerosis: 2 Distinct Entities?

The Journal of Rheumatology ◽

10.3899/jrheum.160736 ◽

2016 ◽

Vol 44 (1) ◽

pp. 40-48 ◽

Cited By ~ 7

Author(s):

Claudio Corallo ◽

Annalisa Santucci ◽

Giulia Bernardini ◽

Natale Figura ◽

Roberto Leoncini ◽

...

Keyword(s):

Systemic Sclerosis ◽

Proteomic Analysis ◽

Type I Collagen ◽

Dermal Fibroblasts ◽

Type I ◽

Matrix Remodeling ◽

Mrna Levels ◽

Qrt Pcr ◽

Unaffected Skin ◽

Skin Samples

Objective.To identify using proteomic analysis the proteins of altered abundance in the affected and unaffected limited cutaneous systemic sclerosis (lcSSc) skin fibroblasts.Methods.Excision biopsies (3 mm) were obtained from the affected and unaffected skin of 5 patients with lcSSc. Dermal fibroblasts were isolated enzymatically. Two-dimensional gel electrophoresis was used to separate and define proteins in affected and unaffected fibroblast lysates. Proteins of altered abundance were identified by mass spectrometry. Differences among skin samples were confirmed also by immunohistochemistry (IHC) and by quantitative real-time PCR (qRT-PCR) for type I collagen (Col-1) and vimentin (VIM).Results.Proteomic analysis revealed different expressions of proteins involved in cytoskeleton organization (27%), extracellular matrix remodeling (11%), response to oxidative stress (22%), energy metabolism (19%), protein metabolism (5%), cellular homeostasis (5%), signal transduction (3%), and protein transcription, synthesis, and turnover (8%). IHC analysis showed that SSc-affected epidermis is thickened and the dermis is strongly reactive to Col-1 and VIM (typical markers of activated myofibroblasts) compared to SSc-unaffected skin, whose stainings are comparable to those of control healthy skin. Overexpression of Col-1 and VIM mRNA levels in affected lcSSc fibroblasts compared to unaffected lcSSc ones was confirmed by qRT-PCR.Conclusion.Consistent with previous studies, these findings are important for 2 reasons: first, because they reveal the opposite behavior of dermal fibroblasts in the unaffected and affected skin areas of the same patient with lcSSc; second, because they demonstrate the histological/histochemical similarities between unaffected skin from patients with lcSSc and healthy control skin.

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Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618297114 ◽

2017 ◽

Vol 114 (14) ◽

pp. E2852-E2861 ◽

Cited By ~ 20

Author(s):

Cunxi Li ◽

Bhuminder Singh ◽

Ramona Graves-Deal ◽

Haiting Ma ◽

Alina Starchenko ◽

...

Keyword(s):

Colorectal Cancer ◽

Type I Collagen ◽

Egf Receptor ◽

Kinase Inhibitor ◽

Single Cells ◽

Three Dimensional ◽

Type I ◽

Strongly Correlated ◽

Human Colorectal Cancer ◽

Neutralizing Monoclonal Antibody

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

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