Γ2 Chain Research Articles

Identification of laminin α5 short arm peptides active for endothelial cell attachment and tube formation.

Laminin-511, a major component of endothelial basement membrane, consists of α5, β1, and γ1 chains. The short arm region of the α5 chain is a structural feature of endothelial laminins. In this study, we identified active sequences for human umbilical vein endothelial cells (HUVECs) using recombinant proteins and synthetic peptides. The short arm of the α5 chain contains three globular domains [laminin N-terminal globular domain, laminin 4 domain a, and laminin 4 domain b (LN, L4a, and L4b)] and three rod-like elements [laminin epidermal growth factor-like domain a, b, and c (LEa, LEb, and LEc)]. The cell attachment assay using recombinant proteins showed that RGD-independent cell attachment sites were localized in the α5LN-LEa domain. Further, we synthesized 70 peptides covering the amino acid sequences of the α5LN-LEa domain. Of the 70 peptides, A5-16 (mouse laminin α5 230-243: LENGEIVVSLVNGR) potently exhibited endothelial cell attachment activity. An active sequence analysis using N-terminally and C-terminally truncated A5-16 peptides showed that the nine-amino acid sequence IVVSLVNGR was critical for the endothelial cell attachment activity. Cell adhesion to the peptides was dependent on both cations and heparan sulfate. Further, the A5-16 peptide inhibited the capillary-like tube formation of HUVECs with the cells forming small clumps with short tubes. The eight-amino acid sequence EIVVSLVN in the A5-16 peptide was critical to inhibit HUVEC tube formation. This amino acid sequence could be useful for grafts and thus modulate endothelial cell behavior for vascular surgery. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Crystal Structure of the Heterotrimeric Integrin-Binding Region of Laminin-111

SummaryLaminins are cell-adhesive glycoproteins that are essential for basement membrane assembly and function. Integrins are important laminin receptors, but their binding site on the heterotrimeric laminins is poorly defined structurally. We report the crystal structure at 2.13 Å resolution of a minimal integrin-binding fragment of mouse laminin-111, consisting of ∼50 residues of α1β1γ1 coiled coil and the first three laminin G-like (LG) domains of the α1 chain. The LG domains adopt a triangular arrangement, with the C terminus of the coiled coil situated between LG1 and LG2. The critical integrin-binding glutamic acid residue in the γ1 chain tail is surface exposed and predicted to bind to the metal ion-dependent adhesion site in the integrin β1 subunit. Additional contacts to the integrin are likely to be made by the LG1 and LG2 surfaces adjacent to the γ1 chain tail, which are notably conserved and free of obstructing glycans.

Vascular Invasion and Stromal S100A4 Expression at the Invasive Front of Colorectal Cancer are Novel Determinants and Tumor Prognostic Markers.

Object: The aim of the present study was to investigate the clinicopathological characteristics and prognostic factors associated with sporadic colorectal cancer (CRC). We examined the clinicopathological findings and immunohistochemical expression of tumor prognostic markers at tumor budding sites to determine their predictive value for patient prognosis.Materials and Methods: Immunohistochemical examination was performed by tissue microarray (TMA) of specimens from 106 patients with CRC. On hematoxylin and eosin (H&E)-stained tumor tissue slides, a representative area of tumor budding at the invasive front was selected for the construction of a TMA. Immunostaining for matrix metalloproteinase-7 (MMP7), the laminin-5 (ln-5) γ2 chain and S100A4 was performed to determine the association between patient survival and these markers.Results: Clinicopathological variables were also assessed. Tumor location, histological type, degree of lymphatic invasion and vascular invasion, tumor stage, epithelial expression of S100A4, stromal cell expression of S100A4 and expression of the ln-5γ2 chain were associated with an increased risk of mortality. Five factors were retained in the multivariate logistic regression analysis. Specifically, the tumor location, degree of lymphatic invasion and vascular invasion, tumor stage and stromal cell expression of S100A4 remained significant predictors of patient survival after controlling for the other variables.Conclusion: Vascular invasion and stromal expression of S100A4 in the tumor budding areas correlated with patient survival. Stromal immunostaining of S100A4 may be useful for identifying high-risk patients with advanced CRC.

Overexpression of LAMC1 predicts poor prognosis and enhances tumor cell invasion and migration in hepatocellular carcinoma.

LAMC1 encodes an extracellular matrix protein, laminin γ1 chain, which is involved in several biological and pathological processes including tissue development, tumor cell invasion and metastasis. In present study, we demonstrated that both LAMC1 protein and mRNA levels were elevated in HCC tissue samples compared with non-cancerous tissue samples according to western blot analyses, immunohistochemistry (IHC) and microarray. Moreover, high LAMC1 expression was positively correlated with incomplete encapsulation (p=0.014), poor overall (OS, p=0.02) and disease-free survival (DFS, p=0.014). Using cell lines, we demonstrated that the levels of LAMC1 mRNA and protein were significantly higher in HCC cell lines than that in LO2 cell line. After the expression of LAMC1 was depressed by siRNA technique, the cell proliferation, migration and invasion were depressed significantly. Taken together, these data suggest that LAMC1 is enriched in HCC; overexpression of LAMC1 predicts poor prognosis, and enhances tumor cell invasion and migration. LAMC1 might be a new biomarker predictive of HCC prognosis and might also be a useful treatment target.

Highly sensitive detection of invasive lung cancer cells by novel antibody against amino-terminal domain of laminin γ2 chain.

The laminin γ2 chain, a subunit of laminin‐332 (α3β3γ2), is a molecular marker for invasive cancer cells, but its pathological roles in tumor progression remain to be clarified. It was recently found that the most N‐terminal, domain V (dV) of γ2 chain has activities to bind CD44 and stimulate tumor cell migration and vascular permeability. In the present study, we prepared a mAb recognizing γ2 dV. Immunoblotting with this antibody, for the first time, showed that proteolytic fragments containing dV in a range of 15–80 kDa were highly produced in various human cancer cell lines and lung cancer tissues. In immunohistochemistry of adenocarcinomas and squamous cell carcinomas of the lung, this antibody immunostained the cytoplasm of invasive tumor cells and adjacent stroma much more strongly than a widely used antibody recognizing the C‐terminal core part of the processed γ2 chain. This suggests that the dV fragments are highly accumulated in tumor cells and stroma compared to the processed γ2 protein. The strong tumor cell staining with the dV antibody correlated with the tumor malignancy grade. We also found that the laminin β3 and α3 chains were frequently overexpressed in tumor cells and tumor stroma, respectively. The cytoplasmic dV detection was especially prominent in tumor cells infiltrating stroma, but low in the cells surrounded by basement membranes, suggesting that the active tumor–stroma interaction is critical for the aberrant γ2 expression. The present study suggests important roles of laminin γ2 N‐terminal fragments in tumor progression.

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes.

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.

22 - Identifying Nitration Sites in Extracellular Matrix Protein Laminin by LC-MS/MS

Reactive nitrogen species (RNS), such as peroxynitrous acid (ONOOH), produced by activated leukocytes and other cells, can nitrate Tyr and Trp residues on proteins to give 3-nitroTyr and 6-nitroTrp. These long-lived chemically-stable post-translational modifications are used as biomarkers of damage, and may contribute to human pathologies. Extracellular matrix (ECM) proteins are highly abundant, long-lived, contain few alternative targets for RNS (Cys and Met), and are exposed to high RNS fluxes. ECM proteins are therefore likely to accumulate nitration products. Evidence has been presented for tyrosine nitration in human atherosclerotic lesions, and we have shown that this co-localizes with the major ECM protein laminin. Furthermore, nitration alters laminin self-assembly and polymerization, and decreases cell binding. Study aim To identify sites of nitration in laminin using tandem mass spectrometry to understand how and why nitration alters laminin structure and function. Results Using standard shotgun proteomics 101 nitration sites have been identified in murine laminin-111 treated with high (500-fold) molar excesses of ONOOH in vitro. These sites include those involved in cell attachment, domains involved in laminin polymerization, and binding sites of other ECM proteins. 20 nitration sites were identified with low (10-fold molar excess) ONOOH concentrations, including Y145 in the laminin γ1 chain that is located in a cell-binding motif, consistent with reduced endothelial cell binding to modified laminin. Conclusions These data indicate that laminin is readily nitrated by low levels of ONOOH, and that this occurs in functionally important domains, consistent with the observed biological changes. Laminin nitration appears to be a sensitive marker of damage, and may contribute to human disease through decreased cell adhesion and altered ECM structure and assembly.

Abstract C05: Pulmonary laminin 332 in tumor cell migration and breast cancer survival

Abstract Metastasis to the lung often leads to the demise of the patient, thus a greater understanding of the process might lead to strategies for better cancer control. Tumor cell metastatic ability is determined by both intrinsic properties of tumor cells and contributions from the microenvironment. The goal of this study was to determine the role of the extracellular matrix protein laminin 332 (LN332) in breast cancer progression. Because tumor cell motility is a requirement for metastasis, we hypothesize that lung tissue harbors substances that induce tumor cell migration. In order to better characterize the interaction of breast carcinoma cells and lung tissue, MCF-7 breast carcinoma cells were added to slides of frozen sections of lung tissue. MCF-7 cells in contact with the lung tissue exhibited properties associated with tumor cell migration, including pseudopodia and lamellipodia. In contrast, MCF-7 cells on glass without lung tissue had smooth borders. Similarly, MCF-7 cells showed migratory properties when co-cultured with small airway epithelial cells (SAEC). LN332 is a component of the basement membrane of lung epithelium, and is thought to induce the migration of several tumor cell types. Immunoblotting revealed all three chains of LN332 in SAEC conditioned medium. To confirm that LN332 was among the components of the lung cells that induced the migration of the breast carcinoma cells, siRNA knockdown of the α3, β3 and γ2 chains of LN332 was performed on SAEC. The conditioned media from knock down and control SAEC was collected for motility assays. Knockdown of the α3 chain alone was sufficient to decrease the expression of the other two chains, but the greatest knockdown of LN332 protein was achieved with simultaneous use of siRNA against the α3, β3 and γ2 chains combined. Conditioned medium from cells with knockdown of LN332 induced tumor cell migration to a lesser extent than conditioned medium from control cells. Taken together, these in vitro data provide evidence that LN332 in the microenvironment has the potential to promote the invasion of metastatic foci into the pulmonary parenchyma. Next, we sought to determine whether LN332 expressed in breast carcinoma cells was associated with any particular phenotype of the tumors, or were associated with tumor prognosis. Tissue microarrays of breast carcinomas excised in 1994 and 1995 from 312 patients were stained for estrogen receptor, progesterone receptor, HER2 and the LN332 β3 chain by immunohistochemistry. LN332 expression was greatest in triple negative and HER2 expressing breast carcinomas. Follow-up for over 20 years on this cohort revealed that patients with tumors expressing LN332 β3 chain had a worse prognosis than patients without LN332 β3 chain in their tumors. Taken together, these studies provide evidence that LN332 in either tumor cells or the microenvironment may be implicated in breast cancer progression. Citation Format: Philip M. Carpenter, Priyanka Sivadas, Argyrios Ziogas, Hoda Anton-Culver. Pulmonary laminin 332 in tumor cell migration and breast cancer survival. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C05.

Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2.

Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.

Deletion of the epidermis derived laminin γ1 chain leads to defects in the regulation of late hair morphogenesis

Laminins are the most abundant non-collagenous basement membrane (BM) components, composed of an α, β and γ chain. The laminin γ1 chain, encoded by LAMC1, is the most abundant γ chain. The main laminin isoforms in the dermo-epidermal junction (DEJ) are laminin-332, laminin-511 and laminin-211, the latter being restricted to the lower part of hair follicles (HFs). Complete deletion of LAMC1 results in lethality around embryonic day 5.5. To study the function of laminin γ1 containing isoforms in skin development and maturation after birth, we generated mice lacking LAMC1 expression in basal keratinocytes (LAMC1EKO) using the keratin 14 (K14) Cre/loxP system. This deletion resulted in loss of keratinocyte derived laminin-511 and in deposition of fibroblast derived laminin-211 throughout the whole DEJ. The DEJ in areas between hemidesmosomes was thickened, whereas hemidesmosome morphology was normal. Most strikingly, LAMC1EKO mice showed delayed HF morphogenesis accompanied by reduced proliferation of hair matrix cells and impaired differentiation of hair shafts (HS). However, this deletion did not interfere with early HF development, since placode numbers and embryonic hair germ formation were not affected. Microarray analysis of skin revealed down regulation of mainly different hair keratins. This is due to reduced expression of transcription factors such as HoxC13, FoxN1, FoxQ1 and Msx2, known to regulate expression of hair keratins. While the role of laminin-511 in signaling during early hair germ formation and elongation phase has been described, we here demonstrate that epidermal laminin-511 is also a key regulator for later hair development and HS differentiation.

What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?

Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell–cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hiPSC culture.

Laminin γ2 knockout mice rescued with the human protein exhibit enamel maturation defects

The epithelial ameloblasts are separated from the maturing enamel by an atypical basement membrane (BM) that is enriched in laminin 332 (LM-332). This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BMs and influences various epithelial cell processes including cell adhesion and differentiation. Mouse models that lack expression of individual LM-332 chains die shortly after birth. The lethal phenotype of laminin γ2 knockout mice can be rescued by human laminin γ2 (LAMC2) expressed using a doxycycline-inducible (Tet-on) cytokeratin 14 promoter-rtTA. These otherwise normal-looking rescued mice exhibit white spot lesions on incisors. We therefore investigated the effect of rescue with human LAMC2 on enamel maturation and structuring of the atypical BM. The maturation stage enamel organ in transgenic mice was severely altered as compared to wild type controls, a structured BM was no longer discernible, dystrophic matrix appeared in the maturing enamel layer, and there was residual enamel matrix late into the maturation stage. Microtomographic scans revealed excessive wear of occlusal surfaces on molars, chipping of enamel on incisor tips, and hypomineralization of the enamel layer. No structural alterations were observed at other epithelial sites, such as skin, palate and tongue. These results indicate that while this humanized mouse model is capable of rescue in various epithelial tissues, it is unable to sustain structuring of a proper BM at the interface between ameloblasts and maturing enamel. This failure may be related to the atypical composition of the BM in the maturation stage and reaffirms that the atypical BM is essential for enamel maturation.

Current approaches to the morphologic diagnosis of different types of congenital epidermolysis bullosa

to optimize the morphological diagnosis of different types of CEB. 28 skin biopsies from 14 patients with different types of CEB were investigated. The investigators performed routine histological examination of skin fragments taken from a bullous area and immunofluorescence antigen mapping using the indirect immunofluorescence test (IIFT) with antibodies against structural proteins of the dermal-epidermal junction (laminin α3, β3, and γ2 chains, keratins 5 and 14, types VII and XVII collagen, α6 and β4 integrin subunits, desmoplakin, plectin, kindlin-1, and plakophillin) of the apparently unaffected skin. The intact skin of healthy individuals, which had been obtained during cosmetic operations, was used as controls in IIFT. Immunofluorescence antigen mapping could determine the type of CEB in all cases and in 86% of cases identify the protein, the impaired production of which was responsible for the development of the disease. Immunofluorescence antigen mapping is an integral part of the comprehensive morphological diagnosis of CEB, acting as an intermediate between the morphological verification of CEB diagnosis and the targeted search for mutations by a molecular genetic method.

Novel Acellular Scaffold Made from Decellularized Schwann Cell Sheets for Peripheral Nerve Regeneration.

Peripheral nerve injuries present a serious clinical need with approximately 50 % of surgical cases achieving only some restoration of function. In order to better guide regenerating nerves, supporting cells of the nerve tissue were grown into sheets and subsequently decellularized, leaving a myriad of surrounding protein as a scaffold. Constructs have been shown to support cell growth and neurite extension in vitro. Future projects will combine various cell types present in the nerve tissue as well as stem cells to fully support and reconstruct architecture of the peripheral nerves.

High-mobility group box 1 promotes extracellular matrix synthesis and wound repair in human bronchial epithelial cells.

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) protein that binds Toll-like receptors (e.g., TLR4) and the receptor for advanced glycated end products (RAGE). The direct effects of HMGB1 on airway structural cells are not fully known. As epithelial cell responses are fundamental drivers of asthma, including abnormal repair-restitution linked to changes in extracellular matrix (ECM) synthesis, we tested the hypothesis that HMGB1 promotes bronchial epithelial cell wound repair via TLR4 and/or RAGE signaling that regulates ECM (fibronectin and the γ2-chain of laminin-5) and integrin protein abundance. To assess impact of HMGB1 we used molecular and pharmacological inhibitors of RAGE or TLR4 signaling in scratch wound, immunofluorescence, and immunoblotting assays to assess wound repair, ECM synthesis, and phosphorylation of intracellular signaling. HMGB1 increased wound closure, and this effect was attenuated by blocking RAGE and TLR4 signaling. HMGB1-induced fibronectin and laminin-5 (γ2 chain) was diminished by blocking RAGE and/or blunting TLR4 signaling. Similarly, induction of α3-integrin receptor for fibronectin and laminin-5 was also diminished by blocking TLR4 signaling and RAGE. Lastly, rapid and/or sustained phosphorylation of SMAD2, ERK1/2, and JNK signaling modulated HMGB1-induced wound closure. Our findings suggest a role for HMGB1 in human airway epithelial cell repair and restitution via multiple pathways mediated by TLR4 and RAGE that underpin increased ECM synthesis and modulation of cell-matrix adhesion.

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

Overexpression of β3 Chains of Laminin-332 is Associated With Clinicopathologic Features and Decreased Survival in Patients With Pancreatic Adenocarcinoma.

Background:Laminin-332 (LM-332, formerly termed laminin-5) is a heterotrimeric glycoprotein that promotes cellular adhesion and migration. The heterotrimer consists of an α3, a β3, and a γ2 chain. The aim of this investigation was to clarify the clinicopathologic significance of laminin-332β3 (LNβ3) chain expression and determine its influence on survival in pancreatic ductal adenocarcinoma.Materials and Methods:Quantitative real-time polymerase chain reaction was used to validate and detect the expression of LNβ3 mRNA in 37 pancreatic carcinoma tissue specimens and non-neoplastic pancreatic tissue samples. In addition, the protein expression of LNβ3 was detected by immunohistochemistry methods in 96 pancreatic carcinoma specimens and 90 non-neoplastic pancreatic tissues. We analyzed the association between immunohistochemically detected LNβ3 expression in pancreatic ductal adenocarcinoma and clinicopathologic characteristics. Survival curves were completed using the Kaplan-Meier method and compared using log-rank analysis.Results:Quantitative real-time polymerase chain reaction indicated that the relative value of LNβ3 mRNA was 1.427±1.554 and 1.423±1.439 by 2−Δ(ΔCt) in pancreatic carcinoma and non-neoplastic pancreatic tissues, respectively, values that were not statistically associated (P=0.991). Immunostaining for LNβ3 was expressed in all patients with pancreatic ductal adenocarcinoma. LNβ3 expression was related to differentiation (P=0.000) and advanced stage (P=0.034). Tumors with low expression of LNβ3 had a survival advantage compared with tumors that had high expression of LNβ3 (P=0.016). Multivariate analysis indicated that location is an independent predictor of overall survival, whereas other clinicopathologic characteristics such as tumor size, duodenal invasion, differentiation, extent of invasion, hepatic metastasis, and expression of LNβ3 were not.Conclusion:Our results suggest that LNβ3 expression may play a key role in the progression and prognosis of pancreatic ductal adenocarcinoma.

Amino-terminal fragments of laminin γ2 chain stimulate migration of metastatic breast cancer cells by interacting with CD44.

Laminin γ2 (Lmγ2) chain, a subunit of the basement membrane protein laminin-332, is regarded as a typical cancer invasion marker. The overexpression of Lmγ2 chain by invasive cancer cells correlates with poor prognosis of cancer patients, and its forced expression in human cancer cells promotes their invasive growth in a nude mouse model. However, its actual roles in cancer progression, as well as the mechanism of its proinvasive effect, remain unclear. CD44 is known to be an important cancer stem cell marker and support cancer progression and stem cell functions. Here we demonstrate that amino-terminal fragments of Lmγ2 interact with CD44 on the membrane of breast cancer cells. Lmγ2 highly bound to the metastatic cell line MDA-MB-231 but poorly to the benign cell line MCF-7. The membrane receptor for Lmγ2 on MDA-MB-231 cells was identified to be the standard form of CD44 (CD44s) by co-immunoprecipitation, affinity chromatography and direct protein interaction assay. Lmγ2 interacted with CD44s through EGF-like repeat 2/3 in the Lmγ2 amino-terminus. Amino-terminal fragments of Lmγ2 induced the phosphorylation of CD44 cytoplasmic domain and stimulated migration of the cancer cells in a CD44-dependent manner. This migration was blocked by inhibitors of TGF-β receptor I (TGF-βRI) kinase. These results suggest that two important tumor markers, Lmγ2 and CD44, cooperate for cancer progression and possibly for cancer stem cell functions. TGF-βRI may be involved in the Lmγ2/CD44 interaction.

Laminin β3 expression as a prognostic factor and a predictive marker of chemoresistance in colorectal cancer.

Laminin-332, a marker of epithelial-mesenchymal transition, is composed of a heterotrimer of α3, β3 and γ2 chains that regulates cell adhesion and migration. This study aimed to disclose the respective clinical significance of laminin β3 immunoexpression in colorectal cancer as a prognostic factor and a predictive marker of chemoresistance. Tissue specimens from 323 Stage II and 232 Stage III colorectal cancer patients who underwent curative resection were assessed using laminin β3 immunostaining. Among Stage III colorectal cancer patients, comparisons of 5-year disease-free survival rates revealed a poorer prognosis for the laminin β3-high group than for the laminin β3-low group (52.3 vs. 70.7%, P = 0.038), while there was no significant difference among Stage II patients. Among laminin β3-low Stage III patients, those who received adjuvant chemotherapy showed marginally better disease-free survival than those who did not receive it (75.8 vs. 62.8%; P = 0.096). Furthermore, multivariate analysis corroborated a distinct benefit of adjuvant chemotherapy in laminin β3-low patients (P = 0.035; hazard risk ratio = 1.66). Analyses of the laminin β3-high group, however, failed to show significance. Laminin β3 chain immunoreactivity was a poor prognostic factor for Stage III colorectal cancer patients, and laminin β3-high patients of Stage III colorectal cancer derived no survival benefit from adjuvant chemotherapy.

Collagen XVII expression correlates with the invasion and metastasis of colorectal cancer.

Collagen XVII has a well-established role as an adhesion molecule and a cell surface receptor located in the type I hemidesmosome of stratified epithelia. Its ectodomain is constitutively shed from the cell surface and suggested to regulate the adhesion, migration, and signaling of cutaneous epithelial cells. Collagen XVII was not previously thought to be expressed by colon epithelial cells. Immunohistochemical analysis of tissue microarray samples of 141 cases of colorectal carcinoma showed that collagen XVII is expressed in normal human colonic mucosa and colorectal carcinoma. In colorectal carcinoma, increased collagen XVII expression was significantly associated with higher TNM stage. It also correlated with infiltrative growth pattern and tumor budding as well as lymph node and distant metastasis. Increased collagen XVII expression was associated with decreased disease-free and cancer-specific survival. Immunofluorescence staining of collagen XVII and its well-known binding partner laminin γ2 chain demonstrated a partial colocalization in normal and tumor tissue. In vitro, the overexpression of murine collagen XVII promoted the invasion of CaCo-2 colon carcinoma cells through Matrigel (BD Biosciences; Bedford, MA). To conclude, this study reports for the first time the expression of collagen XVII in colon epithelium and the association of increased collagen XVII immunoexpression with poor outcome in colorectal carcinoma.