Laminin Γ1 Research Articles

Invasion-Associated Reorganization of Laminin 332 in Oral Squamous Cell Carcinomas: The Role of the Laminin γ2 Chain in Tumor Biology, Diagnosis, and Therapy.

Simple SummaryThe destructive growth of carcinomas is associated with crossing the border between the epithelial and the connective tissue parts of an organ. One component of this borderline, the basement membrane, is the heterotrimeric laminin 332, which mediates the adhesion of basal epithelial cells. This protein, in particular its gamma 2 chain, is fundamentally reorganized during tumor cell invasion. Specific deposition patterns of laminin 332 are also present in oral squamous cell carcinomas and have been shown to be of high diagnostic and predictive value. Furthermore, laminin 332 restructuring is associated with important tumor biological processes, e.g., stromal activation, the development of a motile phenotype, and tumor spreading. In this review, current knowledge in the field is summarized and the recommendation to consider laminin 332 as a promising grading and monitoring parameter and as a potential therapeutic target is discussed.Invasion of the connective tissue by carcinoma cells is accompanied by disintegration and reorganization of the hemidesmosomes, which connect the basement membrane to the basal epithelial cells. In terms of mediating the basement membrane, i.e., basal cell interactions, the heterotrimeric laminin 332 is the most important bridging molecule. Due to this distinct function, laminin 332, especially its gamma 2 chain, came into the focus of cancer research. Specific de novo synthesis and deposition patterns of laminin 332 are evident upon development and progression of oral squamous cell carcinomas (OSCCs). Loss from the basement membrane, cytoplasmic accumulation, and extracellular deposition are associated with crucial processes such as stromal activation and immune response, epithelial to mesenchymal transition, and tumor cell budding. In networks with components of the tumor microenvironment, altered expression of laminin 332 chains, proteolytic processing, and interaction with integrin receptors seem to promote cancer cell migration. Indeed, reorganization patterns are shown to have a high diagnostic and prognostic value. Here, we summarize the current knowledge on laminin 332 reorganization in OSCCs with special focus on its gamma 2 chain and provide, based on the current literature, evidence on its promising role as a grading and monitoring parameter and as a potential therapeutic target.

Activation of Laminin γ2 by Helicobacter pylori Promotes Invasion and Survival of Gastric Cancer Cells With E-Cadherin Defects.

Helicobacter pylori infection induces cellular phenotypes relevant for cancer progression, namely cell motility and invasion. We hypothesized that the extracellular matrix (ECM) could be involved in these deleterious effects. Microarrays were used to uncover ECM interactors in cells infected with H. pylori. LAMC2, encoding laminin γ2, was selected as a candidate gene and its expression was assessed in vitro and in vivo. The role of LAMC2 was investigated by small interference RNA (siRNA) combined with a set of functional assays. Laminin γ2 and E-cadherin expression patterns were evaluated in gastric cancer cases. Laminin γ2 was found significantly overexpressed in gastric cancer cells infected with H. pylori. This finding was validated in vitro by infection with clinical isolates and in vivo by using gastric biopsies of infected and noninfected individuals. We showed that laminin γ2 overexpression is dependent on the bacterial type IV secretion system and on the CagA. Functionally, laminin γ2 promotes cell invasion and resistance to apoptosis, through modulation of Src, JNK, and AKT activity. These effects were abrogated in cells with functional E-cadherin. These data highlight laminin γ2 and its downstream effectors as potential therapeutic targets, and the value of H. pylori eradication to delay gastric cancer onset and progression.

P53 positively regulates the proliferation of hepatic progenitor cells promoted by laminin-521

Hepatic progenitor cells (HPCs) hold tremendous potential for liver regeneration, but their well-known limitation of proliferation hampers their broader use. There is evidence that laminin is required for the proliferation of HPCs, but the laminin isoform that plays the dominant role and the key intracellular downstream targets that mediate the regulation of HPC proliferation have yet to be determined. Here we showed that p53 expression increased gradually and reached maximal levels around 8 days when laminin α4, α5, β2, β1, and γ1 subunit levels also reached a maximum during HPC activation and expansion. Laminin-521 (LN-521) promoted greater proliferation of HPCs than do laminin, matrigel or other laminin isoforms. Inactivation of p53 by PFT-α or Ad-p53V143A inhibited the promotion of proliferation by LN-521. Further complementary MRI and bioluminescence imaging analysis showed that p53 inactivation decreased the proliferation of transplanted HPCs in vivo. p53 was activated by LN-521 through the Integrin α6β1/FAK-Src-Paxillin/Akt axis. Activated p53 was involved in the nuclear translocation of CDK4 and inactivation of Rb by inducing p27Kip1. Taken together, this study identifies LN-521 as an ideal candidate substrate for HPC culture and uncovers an unexpected positive role for p53 in regulating proliferation of HPCs, which makes it a potential target for HPC-based regenerative medicine.

Palmitoyl‑RGD promotes the expression of dermal‑epidermal junction components in HaCaT cells.

With age, the dermal‑epidermal junction (DEJ) becomes thinner and production of its protein components decreases; this may be associated with increased fragility and wrinkling of skin. Topical treatment with palmitoyl‑Arg‑Gly‑Asp (PAL‑RGD) improves facial wrinkles, skin elasticity and dermal density in humans. In the present study, the effect of PAL‑RGD on expression of DEJ components, such as laminin and collagen, was assessed. Human HaCaT keratinocytes were treated with PAL‑RGD. The protein expression levels of laminin‑332, collagenIV and collagenXVII were examined by western blotting. Reverse transcription-quantitative PCR was used to analyze laminin subunit (LAM)A3, LAMB3, LAMC2, collagen typeIV α1 chain (COL4A1) and COL17A1 mRNA expression levels. Western blot analysis showed that the expression levels of proteins comprising the DEJ, including laminin α3, β3 and γ2 and collagenIV and XVII demonstrated a significant dose‑dependent increase following PAL‑RGD treatment. Furthermore, PAL‑RGD treatment significantly enhanced LAMA3, LAMB3, LAMC2, COL4A1 and COL17A1 mRNA expression levels. PAL‑RGD may enhance the DEJ by inducing the expression of laminin‑332, collagenIV and collagenXVII.

Epstein-Barr virus-induced ectopic CD137 expression helps nasopharyngeal carcinoma to escape immune surveillance and enables targeting by chimeric antigen receptors.

Non-keratinizing nasopharyngeal carcinoma (NPC) is a malignancy with a poor prognosis for relapsing patients and those with metastatic disease. Here, we identify a novel disease mechanism of NPC which may be its Achilles' heel that makes it susceptible to immunotherapy. CD137 is a potent costimulatory receptor on activated T cells, and CD137 agonists strongly enhance anti-tumor immune responses. A negative feedback mechanism prevents overstimulation by transferring CD137 from T cells to CD137 ligand (CD137L)-expressing antigen presenting cells (APC) during cognate interaction, upon which the CD137-CD137L complex is internalized and degraded. We found ectopic expression of CD137 on 42 of 122 (34.4%) NPC cases, and that CD137 is induced by the Epstein-Barr virus latent membrane protein (LMP) 1. CD137 expression enables NPC to hijack the inbuilt negative feedback mechanism to downregulate the costimulatory CD137L on APC, facilitating its escape from immune surveillance. Further, the ectopically expressed CD137 signals into NPC cells via the p38-MAPK pathway, and induces the expression of IL-6, IL-8 and Laminin γ2. As much as ectopic CD137 expression may support the growth and spread of NPC, it may be a target for its immunotherapeutic elimination. Natural killer cells that express a CD137-specific chimeric antigen receptor induce death in CD137+ NPC cells, in vitro, and in vivo in a murine xenograft model. These data identify a novel immune escape mechanism of NPC, and lay the foundation for an urgently needed immunotherapeutic approach for NPC.

Abstract WP130: Fibroblast-derived Laminin Promotes Blood-brain Barrier Repair After Stroke Not Through Integrinβ1 Receptors

Blood-brain barrier (BBB) breakdown has been observed in various neurological disorders, including stroke. More importantly, BBB disruption positively correlates with stroke outcome. Laminin, an extracellular matrix protein, plays an important role in the formation and maintenance of blood brain barrier (BBB) integrity under both homeostatic and pathological conditions. Fibroblasts, mainly located in the meninges and around large blood vessels, also secrete laminin. The function of fibroblast-derived laminin in BBB maintenance, however, remains unknown. Here we generated mutant mice with lamininγ1 deficiency in fibroblast-like cells using the Col1a1-Cre line (γ1FKO). These mutants were born at expected Mendelian ratio and failed to show obvious abnormalities, indicating a dispensable role of fibroblast-derived laminin under homeostatic conditions. Next, we examined BBB integrity in these mutants in a collagenase induced hemorrhagic stroke model. Compared to the controls, the mutant mice exhibited aggravated BBB damage and brain injury, including enhanced BBB permeability, elevated inflammatory cell infiltration, reduced tight junction coverage, reduced PDGFRβ coverage, larger hematoma size, worse neurological dysfunction, and increased neuronal cell death. These results suggest that fibroblast-derived laminin exerts a beneficial role in stroke pathogenesis possibly via repairing damaged BBB. Using γ1FKO:Ai14 and Col1α1-Cre+:Ai14 (control) mice, we found significantly reduced fibroblast density in the peri-hematoma region γ1FKO:Ai14 at day7 after injury. Additionally, laminin γ1 knockout in fibroblast cells by CRISPR Cas9 results in reduced fibroblast migration ability. These results suggest that fibroblast-derived laminin play important roles on fibroblast migration. Classical laminin receptors include integrin-α3β1, -α6β1, -α7β1 and -α6β4. To study the receptor regulating laminin- mediated fibroblasts function, we generated integrin β1 mutant mice using Col1a1-Cre line. In these mutants we did not see difference in terms of stoke outcomes as we saw in γ1FKO mice, indicating that integrin β1 is not the receptor regulating laminin- mediated fibroblasts function.

Clear Evidence of LAMA5 Gene Biallelic Truncating Variants Causing Infantile Nephrotic Syndrome.

Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid-resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only seven patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome, and one patient with SRNS with biallelic LAMA5 missense variants. We conducted comprehensive gene screening of Japanese patients with severe proteinuria. With the use of targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in three patients from two families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.[Arg3078*]) and a splice site variant (c.1282 + 1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.[Arg2720*]) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of 8 years, and carried compound heterozygous missense variants (c.1493C>T, p.[Ala498Val] and c.8399G>A, p.[Arg2800His]). Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathologic characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in patients with congenital/infantile nephrotic syndrome.

Abstract PO-089: Identification of a LAMC2-regulated network featuring targetable effectors for dual therapies in pancreatic cancer

Abstract Background: Pancreatic cancer stands as one of the deadliest tumors, in part due to the limited efficacy of currently existing therapies. Pancreatic cancer is characterized at the genomic level by the almost universal presence of KRAS mutations. Phenotypically, it features an incipient desmoplastic stroma with a protein component formed by collagens, fibronectin and laminins. A member of the laminin family, LAMININ γ2 or LAMC2, was previously identified by our group as part of a cross-tumors KRAS signature whose high expression was a marker of poor survival in PDAC patients. While a role for LAMC2 in migration and invasion has been previously described, little is known about a potential function in cell proliferation and viability. Moreover, LAMC2 inhibition has been shown to enhance the activity of conventional chemotherapeutic agents. However, whether its abrogation can increase the effect of targeted therapies is yet to be defined. Finally, the direct relationship between LAMC2 and the KRAS oncogene network is yet to be defined. Methods: A meta-analysis of several human PDAC data sets and survival analysis of the TCGA data were performed to query the expression levels and prognosis significance of LAMC2. Human and mouse PDAC cell lines as well as a mouse model of PDAC (KrasLSLG12D, Tp53f/f, Ptf1aCre) were used to assess LAMC2 expression. In vitro (2D and 3D organoids) and in vivo models derived from human and mouse PDAC cell lines or primary tumors were used to define the functional role of LAMC2 in PDAC. Cellular and molecular analysis were deployed to dissect the mechanism of action of LAMC2 in PDAC. A dual pharmacological combination based on LAMC2-regulated effectors was also tested in human and mouse pancreatic cancer models. Results: At the clinical level, we observed that LAMC2 is overexpressed in human PDAC patients with regard to normal tissue. The overexpression of LAMC2 was recapitulated in human and mouse cell lines. Genetic depletion of LAMC2 had an adverse effect in 2D proliferation, clonogenic efficiency, 3D organoid growth, and PDAC-based xenograft/allograft tumor development. This deleterious effect was driven by an induction in apoptosis and a decrease in S phase consistent across species. Mechanistically, LAMC2 was linked to the KRAS pathway via transcriptional regulation of the transcription factor FOSL1/AP1, which was also a member of the cross-tumors signature. Furthermore, LAMC2 controlled a gene signature that overlaps with KRAS- and FOSL1-regulated gene signatures, which was also highly expressed in various pancreatic cancer data sets, and includes the targetable kinase AXL. Lastly, concomitant pharmacological inhibition of AXL and the FOSL1 upstream regulator MEK1/2 displayed a more adverse effect on human and pancreatic pancreatic cancer than each single drug alone. Conclusion: These data suggest that LAMC2 is a molecular target tightly linked to KRAS oncogene signaling that regulates downstream effectors amenable to the development of combination therapies. Citation Format: Shruthi Narayanan, Oihane Erice, Iker Feliu, Caterina Vicentini, Rodrigo Entrialgo-Cadierno, Karmele Valencia, Elisabet Guruceaga, Purvesh Khatri, Vicenzo Corbo, Silvestre Vicent Cambra, Mariano Ponz-Sarvise. Identification of a LAMC2-regulated network featuring targetable effectors for dual therapies in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-089.

A novel model of nephrotic syndrome results from a point mutation in Lama5 and is modified by genetic background

Nephrotic syndrome is characterized by severe proteinuria, hypoalbuminaemia, edema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a crucial role in slit diaphragm signaling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin β2 and laminin γ1, is a major component of the glomerular basement membrane, a critical component of the glomerular filtration barrier. Mutations in LAMA5 were recently identified in children with nephrotic syndrome. Here, we have identified a novel missense mutation (E884G) in the uncharacterized L4a domain of LAMA5 where homozygous mice develop nephrotic syndrome with severe proteinuria with histological and ultrastructural changes in the glomerulus mimicking the progression seen in most patients. The levels of LAMA5 are reduced in vivo and the assembly of the laminin 521 heterotrimer significantly reduced in vitro. Proteomic analysis of the glomerular extracellular fraction revealed changes in the matrix composition. Importantly, the genetic background of the mice had a significant effect on aspects of disease progression from proteinuria to changes in podocyte morphology. Thus, our novel model will provide insights into pathologic mechanisms of nephrotic syndrome and pathways that influence the response to a dysfunctional glomerular basement membrane that may be important in a range of kidney diseases.

Expression and localization of amelotin, laminin γ2 and odontogenesis-associated phosphoprotein (ODAPH) on the basal lamina and junctional epithelium.

At maturation stage of enamel development, a specialized basal lamina (sBL) was built between ameloblasts and enamel. After the teeth eruption, the ameloblasts transform into the inner cell layer of junctional epithelium. The inner cell layer forms the internal basal lamina of junctional epithelium. However, the composition of the sBL and internal basal lamina was not clarified. The objective of our study was to make a description of the localization of amelotin (AMTN), laminin γ2 (LAMC2) and Odontogenesis-associated phosphoprotein (ODAPH) on the sBL and internal basal lamina. In immunohistochemical study, AMTN, LAMC2 and ODAPH were detected on the sBL at maturation stage. AMTN was also detected in ameloblasts at maturation stage. The expression of AMTN decreased from early-to-late maturation stage. In contrast, the expression of LAMC2 and ODAPH was stable. Immunofluorescence double-staining showed the localization of AMTN was close to enamel surface. However, the localization of ODAPH was close to ameloblasts. LAMC2 and ODAPH were observed on internal basal lamina of junctional epithelium. In contrast, no expression of AMTN was detected on internal basal lamina of junctional epithelium. Our results suggested that ODAPH might participate in enamel maturation and periodontal health, which might provide a better understanding of enamel defects and periodontal disease in clinic.

Variants in LAMC3 Causes Occipital Cortical Malformation.

Occipital cortical malformation (OCCM) is a disease caused by malformations of cortical development characterized by polymicrogyria and pachygyria of the occipital lobes and childhood-onset seizures. The recessive or complex heterozygous variants of the LAMC3 gene are identified as the cause of OCCM. In the present study, we identified novel complex heterozygous variants (c.470G > A and c.4030 + 1G > A) of the LAMC3 gene in a Chinese female with childhood-onset seizures. Cranial magnetic resonance imaging was normal. Functional experiments confirmed that both variant sites caused premature truncation of the laminin γ3 chain. Bioinformatics analysis predicted 10 genes interacted with LAMC3 with an interaction score of 0.4 (P value = 1.0e–16). The proteins encoded by these genes were mainly located in the basement membrane and extracellular matrix component. Furthermore, the biological processes and molecular functions from gene ontology analysis indicated that laminin γ3 chain and related proteins played an important role in structural support and cellular processes through protein-containing complex binding and signaling receptor binding. KEGG pathway enrichment predicted that the LAMC3 gene variant was most likely to participate in the occurrence and development of OCCM through extracellular matrix receptor interaction and PI3K-Akt signaling pathway.

Abstract 1411: LAMC2 promotes progression and gemcitabine resistance through modulation of EMT and ATP-binding transporters in pancreatic ductal adenocarcinoma

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor prognosis. Gemcitabine-based therapeutic options remain the first treatment choice for the majority of PDAC patients. Unfortunately, however, currently there is lack of availability of robust prognostic and predictive biomarkers in patients with PDAC. Laminin γ2 (LAMC2) is frequently overexpressed in several cancers, and its high expression facilitates cancer development and chemoresistance. The role of LAMC2 in disease pathogenesis, as well as its clinical significance, if any, in PDAC remains unclear. Considering that a better understanding of its molecular function will likely help improve the prognosis in PDAC patients, herein, we undertook this study to elucidate the clinical and biological function of LAMC2 in PDAC. Methods: LAMC2 expression was assessed in tissue specimens from a cohort of 114 PDAC patients who received surgical treatment. This was followed by examining the clinical significance of this gene in multiple patient cohorts. The qRT-PCR based gene expression data were subsequently analyzed in the context of prognosis of PDAC patients. Finally, functional characterization of the role of LAMC2 in PDAC was investigated in a series of experiments following small interfering RNA (siRNA)- based knockdown of this gene in pancreatic cancer (PC) cells. Results: Higher expression of LAMC2 significantly correlated with poor survival in PDAC patient cohort (overall survival [OS]: 23.0 vs 14.2 months, Hazard ratio [HR]=1.61; 95% confidence interval [CI], 1.09-2.79, P < 0.01, relapse-free survival [RFS]: 9.0 vs 6.2 months, HR=1.61, 95%CI, 0.96-2.71, P < 0.05). In multivariate analysis which included several clinical factors, elevated LAMC2 expression served as an independent prognostic factor for OS (HR=1.71; 95% CI, 1.09-2.68). Moreover, higher LAMC2 expression levels predicted response to gemcitabine-based adjuvant therapy in patients with PDAC (HR=1.66, 95%CI, 0.94-2.93). The inhibition of LAMC2 expression enhanced gemcitabine sensitivity and induction of apoptosis in PC cell lines. Moreover, siRNA mediated suppression of LAMC2 expression led to inhibited colony formation ability, migration and invasion potential. Furthermore, LAMC2 was found to regulate the expression of various epithelial-mesenchymal transition (EMT) associated genes. In addition, LAMC2 expression significantly correlated with genes associated with the several key ATP-binding cassette (ABC) transporters in both PC cell lines and PDAC tissues. Conclusion: We conclude that LAMC2 regulates gemcitabine sensitivity through EMT and ABC transporter pathways in PDAC, and this gene may be a potential therapeutic target in patients with PDAC. Citation Format: Yasuyuki Okada, Satoshi Nishiwada, Naoki Takahashi, Tetsuji Takayama, Ajay Goel. LAMC2 promotes progression and gemcitabine resistance through modulation of EMT and ATP-binding transporters in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1411.

Oncolytic H-1 parvovirus binds to sialic acid on laminins for cell attachment and entry

H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies.

Serum Laminin γ2 Monomer as a Diagnostic and Predictive Biomarker for Hepatocellular Carcinoma.

Structural dynamics of basement membrane components are still to be elucidated in the process of hepatocarcinogenesis. We evaluated the characteristics of HCC expressing laminin γ2 monomer (LG2m), a basement membrane component not detected in normal tissues, for HCC diagnosis. We further determined whether elevated serum LG2m is a risk factor for HCC development in patients with chronic hepatitis C (CHC). In HCC cell lines, LG2m was expressed in alpha-fetoprotein (AFP)-negative, CD90-positive cells characterized by highly metastatic natures. Using 14 cell lines and 258 HCC microarray data, we identified that LG2m gene signature was associated with Hoshida's S1/Boyault's G3 molecular subclasses with poor prognosis, which could not be recognized by AFP. Serum LG2m was assessed in 24 healthy donors, 133 chronic liver disease patients, and 142 HCC patients, and sensitivity and specificity of LG2m testing for HCC diagnosis were 62.9% and 70.5%, respectively (cutoff, 30pg/mL). We evaluated the consequence of LG2m elevation in two independent HCC cohorts (n=47 and n=81), and LG2m-high HCC showed poor prognosis with later development of distant organ metastasis (cutoff, 60pg/mL). LG2m was slightly elevated in a subset of CHC patients, and Kaplan-Meier analysis indicated a high incidence of HCC (n=70). For validation, we enrolled 399 CHC patients with sustained virological response (SVR) as a multicenter, prospective study, and serum LG2m elevation correlated with a high incidence of HCC in the CHC patients with SVR (P<0.0001). LG2m is a predictive biomarker for the development of metastatic HCC. Elevated serum LG2m is an HCC risk in CHC patients who have achieved SVR.

LAMC2 promotes cancer progression and gemcitabine resistance through modulation of EMT and ATP-binding cassette transporters in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor prognosis. Gemcitabine remains an effective option for the majority of PDAC patients. Unfortunately, currently no reliable prognostic and predictive biomarkers of therapeutic response are available for the patients with PDAC. Laminin γ2 (LAMC2) is overexpressed in several cancers, and its high expression facilitates cancer development and chemoresistance. However, its functional role in PDAC remains unclear, and a better understanding of this will likely help improve the prognosis of PDAC patients. This study aimed to elucidate the clinical and biological role of LAMC2 in PDAC. We first analyzed the expression levels of LAMC2 by real-time reverse transcription PCR in a cohort of 114 PDAC patients. Interestingly, higher expression of LAMC2 significantly correlated with poor survival in PDAC cohort. In addition, elevated LAMC2 expression served as a potential prognostic marker for survival. Subsequently, functional characterization for the role of LAMC2 in PDAC was performed by small interfering RNA knockdown in pancreatic cancer (PC) cell lines. Interestingly, inhibition of LAMC2 in PC cells enhanced the gemcitabine sensitivity and induction of apoptosis. Moreover, it inhibited colony formation ability, migration and invasion potential. Furthermore, LAMC2 regulated the expression of epithelial-mesenchymal transition (EMT) phenotype. In addition, LAMC2 significantly correlated with genes associated with the expression of ATP-binding cassette (ABC) transporters in PC cells and PDAC patients. In conclusion, these results suggest that LAMC2 regulates gemcitabine sensitivity through EMT and ABC transporters in PDAC and may be a novel therapeutic target in PDAC patients.

Identification of laminin γ2 as a prognostic and predictive biomarker for determining response to gemcitabine-based therapy in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. While the extracellular matrix component plays an integral role in PDAC pathogenesis and mediating chemoresistance, its role in predicting response to chemotherapy in patients with PDAC remains unclear. MethodsWe performed a systematic biomarker discovery by analysing genome-wide transcriptomic profiling data from 423 patients (GSE71729, GSE21501 and The Cancer Genome Atlas [TCGA]) for predicting overall survival (OS). This was subsequently validated in two independent clinical cohorts of 270 patients with PDAC (training cohort, n = 121, and validation cohort, n = 149). In addition, we investigated endoscopic ultrasound-fine needle aspiration biopsy specimens from 51 patients with PDAC with an unresectable cancer for predicting therapeutic response to gemcitabine-based therapy. ResultsAfter rigorous bioinformatic analysis, we identified laminin γ2 (LAMC2) to be a significant prognostic factor in all three PDAC data sets (GSE71729: hazard ratio [HR] = 2.04, P = 0.002; GSE21501: HR = 2.17, P = 0.031; TCGA: HR = 2.57, P < 0.001). High LAMC2 expression in patients with PDAC was associated with a significantly poor OS and relapse-free survival in both the training (P < 0.001, P < 0.001) and validation cohorts (P = 0.001, P = 0.026). More importantly, LAMC2 expression robustly identified patients with PDAC and unresectable disease and those who responded to gemcitabine-based therapy (area under the curve = 0.79; 95% confidence interval [CI], 0.65–0.89). The univariate logistic regression analysis revealed that high LAMC2 expression was the only factor that predicted poor response to gemcitabine in patients with PDAC (odds ratio = 4.90; 95% CI, 1.45–16.6; P = 0.011). ConclusionWe conclude that LAMC2 is a novel prognostic and predictive biomarker for gemcitabine-based therapy in both the adjuvant and palliative setting; which could have significant impact on precision and individualised treatment of patients with PDAC.

Effects of laminin-111 peptide coatings on rat neural stem/progenitor cell culture

Neurons require adhesive scaffolds for their growth and differentiation. Laminins are a major cell adhesive component of basement membranes and have various biological activities in the peripheral and central nervous systems. Here, we evaluated the biological activities of 5 peptides derived from laminin-111 as a scaffold for mouse neuroblastoma Neuro2a cells and rat neural stem/progenitor cells (NPCs). The 5 peptides showed Neuro2a cell attachment activity similar to that of poly-d-lysine. However, when NPCs were cultured on the peptides, 2 syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139–150), demonstrated significantly higher cell attachment and neurite extension activities than other peptides including integrin-binding ones. Long-term cell culture experiments showed that both AG73 and C16 supported the growth of neurons and astrocytes that had differentiated from NPCs. Furthermore, C16 markedly promoted the expression of neuronal markers such as synaptosomal-associated protein-25 and syntaxin 1A. These results indicate that AG73 and C16 are useful for NPC cultures and that C16 can be applied to specialized research on synapses in differentiated neurons. These peptides have the potential for use as valuable biomaterials for NPC research.

Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. We examined the role of the basement membrane proteins laminin subunits α3, β3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, β3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

Differential TM4SF5-mediated SIRT1 modulation and metabolic signaling in nonalcoholic steatohepatitis progression.

Nonalcoholic fatty liver disease is a chronic condition involving steatosis, steatohepatitis and fibrosis, and its progression remains unclear. Although the tetraspanin transmembrane 4 L six family member 5 (TM4SF5) is involved in hepatic fibrosis and cancer, its role in nonalcoholic steatohepatitis (NASH) progression is unknown. We investigated the contribution of TM4SF5 to liver pathology using transgenic and KO mice, diet- or drug-treated mice, in vitro primary cells, and in human tissue. TM4SF5-overexpressing mice exhibited nonalcoholic steatosis and NASH in an age-dependent manner. Initially, TM4SF5-positive hepatocytes and liver tissue exhibited lipid accumulation, decreased Sirtuin 1 (SIRT1), increased sterol regulatory-element binding proteins (SREBPs) and inactive STAT3 via suppressor of cytokine signaling (SOCS)1/3 upregulation. In older mice, TM4SF5 promoted inflammatory factor induction, SIRT1 expression and STAT3 activity, but did not change SOCS or SREBP levels, leading to active STAT3-mediated ECM production for NASH progression. A TM4SF5-associated increase in chemokines promoted SIRT1 expression and progression to NASH with fibrosis. Suppression of the chemokine CCL20 reduced immune cell infiltration and ECM production. Liver tissue from high-fat diet- or CCl4 -treated mice and human patients exhibited TM4SF5-dependent steatotic or steatohepatitic livers with links between TM4SF5-mediated SIRT1 modulation and SREBP or SOCS/STAT3 signaling axes. TM4SF5-mediated STAT3 activation in fibrotic NASH livers increased collagen I and laminin γ2. Both collagen I α1 and laminin γ2 suppression resulted in reduced SIRT1 and active STAT3, but no change in SREBP1 or SOCS, and abolished CCl4 -mediated mouse liver damage. TM4SF5-mediated signaling pathways that involve SIRT1, SREBPs and SOCS/STAT3 promoted progression to NASH. Therefore, TM4SF5 and its downstream effectors may be promising therapeutic targets to treat nonalcoholic fatty liver disease. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Integrin subunitβ1 and laminin γ1 chain expression: a potential prognostic biomarker in cervical cancer.

Aim: The aim of this study was to analyze the prognostic value of integrin subunit β1 and laminin γ1 chain in patients with cervical cancer (CC). Materials & methods: The study included 96 samples. Cytological diagnosis, human papillomavirus (HPV)genotyping, HPV integration status and integrin subunit β1 and laminin γ1 chain expressions were performed or determined using Papanicolaou smear, INNO-LiPA® Genotyping Extra Kit, in situ hybridization, and immunocytochemistry, respectively. The association between variables was calculated using chi-squared and Fisher's exact test; logistic regression analysis was performed to calculate odds ratios and CI at 95%. Results: Our results show that integrin subunitβ1and laminin γ1 chain expressions increase according to tumor progression. Integrin subunit β1 and laminin γ1 chain expressions are associated with cytological diagnosis (p <0.001 and p =0.001, respectively) and laminin γ1 chain expression with the integration status of HPV (p <0.001). Moderate/high expressions of integrin subunit β1 and laminin γ1 chain were correlated with overall survival and increased risk of CC (6.86 and 3.75, respectively), the odds ratiowas 12.91 when the moderate/high expression of integrin subunit β1 and laminin γ1 chain were combined. Conclusion: Our results suggest that integrin subunit β1 and laminin γ1 chain expressions could be a prognostic biomarker in CC.