Laminin-binding Integrins Research Articles

Restoring mechanophenotype reverts malignant properties of ECM-enriched vocal fold cancer.

Increased extracellular matrix (ECM) and matrix stiffness promote solid tumor progression. However, mechanotransduction in cancers arising in mechanically active tissues remains underexplored. Here, we report upregulation of multiple ECM components accompanied by tissue stiffening in vocal fold cancer (VFC). We compare non-cancerous (NC) and patient-derived VFC cells - from early (mobile, T1) to advanced-stage (immobile, T3) cancers - revealing an association between VFC progression and cell-surface receptor heterogeneity, reduced laminin-binding integrin cell-cell junction localization and a flocking mode of collective cell motility. Mimicking physiological movement of healthy vocal fold tissue (stretching/vibration), decreases oncogenic nuclear β-catenin and YAP levels in VFC. Multiplex immunohistochemistry of VFC tumors uncovered a correlation between ECM content, nuclear YAP and patient survival, concordant with VFC sensitivity to YAP-TEAD inhibitors in vitro. Our findings present evidence that VFC is a mechanically sensitive malignancy and restoration of tumor mechanophenotype or YAP/TAZ targeting, represents a tractable anti-oncogenic therapeutic avenue for VFC.

Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

BackgroundThe aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell–matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions. As over-expression of the laminin-binding α6 integrin (Itgα6) is associated with poor prognosis and reduced survival in breast cancer, we here investigate the role of Itgα6 in mammary tumorigenesis.MethodsWe used Blg-Cre; Brca1F/F; Trp53F/F mice, a model that phenocopies human basal-like breast cancer with BRCA1 mutations. We generated mutant mice proficient or deficient in Itgα6 expression and followed tumor formation. Mammary tumors and pretumoral tissues were characterized by immunohistochemistry, flow cytometry, RT-qPCR, Western blotting and organoid cultures. Clonogenicity of luminal progenitors from preneoplastic glands was studied in 3D Matrigel cultures.ResultsWe show that Itga6 deletion favors activation of p16 cell cycle inhibitor in the preneoplastic tissue. Subsequently, the amplification of luminal progenitors, the cell of origin of Brca1-deficient tumors, is restrained in Itgα6-deficient gland. In addition, the partial EMT program operating in Brca1/p53-deficient epithelium is attenuated in the absence of Itgα6. As a consequence of these events, mammary tumor formation is delayed in Itgα6-deficient mice. After tumor formation, the lack of Itgα6 does not affect tumor growth but rather alters their differentiation, resulting in reduced expression of basal cell markers.ConclusionsOur data indicate that Itgα6 has a pro-tumorigenic role in Blg-Cre; Brca1F/F; Trp53F/F mice developing basal-like mammary tumors. In particular, we reveal that Itgα6 is required for the luminal progenitor expansion and the aberrant partial EMT program that precedes the formation of BRCA1 deficient tumors.

Abstract 4618: PIM kinases drive cell adhesion mediated drug resistance in prostate cancer

Abstract Introduction: Castration-resistant prostate cancer (CRPC) is an incurable and lethal progression of the disease that does not respond to any treatment options. Cell adhesion to the extracellular matrix (ECM) activates survival pathways, allowing cancer cells to evade anti-cancer therapy, a phenomenon known as cell adhesion-mediated drug resistance. Bone, which is the primary site of prostate cancer metastasis, is known to be enriched in laminin, a ubiquitous ECM protein. In addition, laminin binding integrins, α6β1 and α3β1, are the major integrins expressed in CRPC, suggesting that laminin-dependent adhesion provides a survival advantage. Based on these findings, activation of the laminin-binding integrin, α6β1, is an intriguing factor mediating resistance to therapy in CRPC. PIM1 is an oncogenic Ser/Thr kinase that is elevated in CRPC and is known to promote resistance to therapy. In contrast, to many pro-survival kinases, PIM kinases do not possess any obvious regulatory domains. Current evidence suggests that PIM1 is constitutively active when it is expressed, so PIM1 expression level directly correlates with its catalytic activity. Despite this fact, little is known about the internal and external mechanisms that dictate PIM1 protein levels and spatial activation in cancer cells. Here, we identify PIM1 as a key survival signal that contributes to cell adhesion-mediated drug resistance. Experimental procedures: Supporting the importance of this signaling axis in human tumors, multiplex IHC of a TMA containing prostate adenocarcinoma and CRPC bone metastases to show that integrin α6 and PIM1 are coexpressed and spatially correlated in primary and metastatic prostate cancer. To examine the spatiotemporal dynamics of PIM1 during cell adhesion, We developed a kinase activity reporter (PIMKAR) to monitor PIM catalytic activity in live cells. Because we observed PIM was highly localized to the membrane in patient tumors, we tagged PIMKAR with a myristylation sequence to specifically measure PIM activity at the membrane vs cytosol. Immunofluorescence and biochemical techniques were used to demonstrate the effect of PIM1 on mitophagy and oxidative stress. Conclusions: We demonstrate that activation of integrin α6β1 downstream of cell adhesion stabilizes PIM1 by blocking its proteasomal degradation. The resulting upregulation of PIM1 serves to reduce oxidative stress through complementary mechanisms reducing mitochondrial fragmentation and increasing the removal of damaged mitochondria by increasing mitophagy. Mechanistically, PIM1 upregulates BNIP3 to enhance mitophagy and thereby reduce oxidative stress in the cell. Together, these results expand our understanding of the mechanisms regulating PIM1 activation and provide evidence for the use of PIM inhibitors in bone mCRPC and other cancers where laminin is a major component of the ECM Citation Format: Caitlyn Flores, Shailender Chauhan, Cynthia Miranti, Anne Cress, Gregory Rogers, Noel Warfel. PIM kinases drive cell adhesion mediated drug resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4618.

Abstract 4206: Regional transcriptomic profiling of the head and neck squamous cell carcinoma tumor microenvironment paired with murine models identify extracellular matrix components that mediate tumor progression and immune suppression

Abstract Purpose: A substantial contribution to the poor prognosis for head and neck squamous cell carcinoma (HNSCC) patients is the progression to distant metastasis. This occurs in 30% of patients over the course of their disease, and radically reduces the 5-year survival rate to under 50%. Investigations in recent years have begun to unveil the intricate interplay of the extracellular matrix (ECM) in both tumor-intrinsic signaling and immunoregulation. However, studies conducted in the context of metastatic have been limited. Here, we employ spatial transcriptomics on HNSCC patient biopsies to identify ECM components that may contribute to cancer progression and immune suppression. Experimental design: Comprehensive transcriptome profiling of HNSCC patient biopsies was conducted using the GeoMx Digital Spatial Profiler (DSP). This approach allowed us to characterize regions of the tumor microenvironment (TME) by immunofluorescent staining for subsequent bioinformatic analysis. To investigate the biological consequences of associations observed in our dataset, we performed in vivo murine studies. We have produced a series of SCC C57BL/6 murine cell lines derived from Smad4-/-KrasG12D Keratin15+ stem cells, which faithfully mimic the clinical progression of HNSCC tumors, including metastasis. Using these murine models, we applied relevant pharmacologic inhibitors and genetic knockdown of targets identified with our GeoMx DSP analysis. In vitro immune-based and functional assays were conducted to elucidate the underlying mechanisms contributing to SCC progression. Results: GeoMx DSP analysis revealed that laminins were enriched in stage IV HNSCC patient stroma, relative to lower stage patients. This coincided with TGFβ enrichment and inversely associated with signatures of anti-tumor immune infiltration. Analysis of The Cancer Genome Atlas data revealed similar inverse correlations in SCCs, distinct from breast cancer and lung adenocarcinoma. Our metastatic SCC murine lines likewise exhibited elevated levels of these laminin proteins, accompanied by high levels of laminin-binding integrins and TGFβ-Smad signaling. Independent of impacting cell proliferation, shRNA knockdown of laminin decreased tumor volume in an immune-competent setting. Targeting laminin-integrin signaling with the small molecule inhibitors Galunisertnib and Buparlisib also reduced the metastatic capacity of these cells in C57BL/6 mice. Conclusions: Spatial transcriptomic profiling of the HNSCC TME uncovers intricate associations and promising targets not readily discernible in bulk RNAseq analyses. Validation through genetic and pharmacological approaches in immune-competent models underscores their potential as strategies to reduce HNSCC burden. Citation Format: John D. Aleman, Khoa A. Nguyen, Erica Wong, Yao Ke, Hanne T. Lind, Clifford G. Tepper, Andrew C. Birkeland, Christian D. Young, Sana D. Karam, Xiao-Jing Wang. Regional transcriptomic profiling of the head and neck squamous cell carcinoma tumor microenvironment paired with murine models identify extracellular matrix components that mediate tumor progression and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4206.

Biophysical phenotype mixtures reveal advantages for tumor muscle invasion in vivo

Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6β1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.

Convection and extracellular matrix binding control interstitial transport of extracellular vesicles.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3β1 and α6β1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75μm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3β1 and α6β1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.

Abstract 3834: Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma

Abstract Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) have a dismal survival rate. The effects of the extracellular matrix (ECM) on cancer progression have been long studied, but the roles of specific integrins in the process of HNSCC metastasis remain to be dissected. This study aims to determine how HNSCC cells affect the production of laminin-binding integrins, and how these integrins participate in the ECM interactions necessary for a metastatic phenotype. Experimental design: Our laboratory has produced syngeneic mouse SCC cell lines, P029 and A223, derived from Keratin15+ stem cells with Smad4 loss and KrasG12D mutation. In syngeneic recipients, SCCs derived from P029 cells transplanted to the flank mouse skin produced spontaneous metastases to the lung while SCCs derived from A223 cells did not form metastases. Having the same genotype, these cell lines serve as models to examine cancer cell interactions with the ECM and resulting effects on invasion and metastasis. Bulk RNA sequencing (RNAseq) was performed to compare cultured metastatic P029 cells versus non-metastatic A223 cells and identify differentially expressed genes that regulate SCC cells and ECM interactions. Immunoassays and functional invasion assays were performed to evaluate ECM-cancer cell signaling and influence on cancer cell invasion in these two cell lines. Results: RNAseq analysis revealed that, relative to A223 cells, P029 cells have increased levels of integrins, the main mechanoreceptors for numerous ECM ligands and matrix proteins. Laminin-binding integrins, including integrins α4 and β6, were greater at the RNA and protein levels. Additionally, P029 cells expressed higher levels of fibronectin and laminin coding genes compared to non-metastatic A223 cells. Correlatively, high expression of these matrix proteins is associated with worse patient survival. Additionally, P029 SCC cells displayed differential gene expression of tight and apical junctions, PI3K signaling components, and regulators of cytoskeletal remodeling. Furthermore, ELISA and western blot analysis revealed that P029 has aberrant TGFβ-Smad signaling as indicated by the elevated release of TGFβ-1 protein and higher levels of phosphorylated Smad2 and Smad3 relative to non-metastatic A223. Treating P029 cells with TGFβ-1 significantly increased their motility and invasion. Conversely, migration and motility of P029 cells were radically reduced by the TGFβ inhibitor galunisertib. Additionally, the mouse cytokine array revealed that mouse plasma bearing P029 tumors had a greater circulation of CXCL16, MMP-9, proliferin, and serpin E1. These proteins are associated with ECM remodeling and metastasis in HNSCC. Conclusions: SCC cells with metastatic properties upregulate integrins which bind to non-collagen matrix proteins and have elevated invasive and migratory capacity contributed from activated TGFβ signaling. Citation Format: John Aleman, Khoa A. Nguyen, Yao Ke, Christian D. Young, Xiao-Jing Wang. Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3834.

Abstract 3835: Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia

Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) migration, invasion, metastasis, and decreased cancer-specific survival. Kindlin-2 (FERMT2) is a β1 integrin adaptor and mechanosensory focal adhesion (FA) protein that activates and clusters integrins in response to structural ECM alterations in the tumor microenvironment. Our goal was to determine if integrin-kindlin-2 adhesion complexes (kindlin-2:α6β1) were responsive to hypoxia, a physiologically relevant and altered microenvironment in PCa progression. Five different endpoints were tested including the biochemical analysis of kindlin-2 complexes, qRT-PCR, immunoblotting, immunocytochemistry, and electric cell impedance sensing (ECIS). Using DU145 prostate cancer cells grown under hypoxia (1% O2) for up to 16 hours, the results showed a reversible increase in kindlin-2:α6β1 complexes with maximal assembly within 4 hours and disassembly starting by 8 hours. Notably, kindlin-2:α6β1 complexes were found exclusively within membrane projections and were not observed within hypoxia-inducible paxillin (PXN)-containing FAs. The hypoxia induced kindlin-2:α6β1 complexes and classical FAs were dependent on kindlin-2 as determined by CRISPR-Cas9 heterozygous deletion of FERMT2. Protein co-localization of α6 integrin and PXN with kindlin-2 within membrane projections and FAs, respectively, was also induced under hypoxia. Further, non-invasive ECIS measurements in live cells confirmed functional cell-cell and cell-ECM dynamics driven by hypoxia and requiring kindlin-2. Our results indicate that the kindlin-2:α6β1 complexes are uniquely associated with FA-independent membrane projections induced by hypoxia, a tumor microenvironment associated with aggressive prostate cancer. The novel kindlin-2:α6β1 complexes may represent an actionable pharmacological target for blocking escape of organ confined disease and metastasis promoting steps of human prostate cancer. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Jaime M.C. Gard, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3835.

Revisiting laminin and extracellular matrix remodeling in metastatic squamous cell carcinoma: What have we learned after more than four decades of research?

Patients with squamous cell carcinoma (SCC) have significantly lower survival upon the development of distant metastases. The extracellular matrix (ECM) is a consistent yet dynamic influence on the metastatic capacity of SCCs. The ECM encompasses a milieu of structural proteins, signaling molecules, and enzymes. Just over 40 years ago, the fibrous ECM glycoprotein laminin was identified. Roughly four decades of research have revealed a pivotal role of laminins in metastasis. However, trends in ECM alterations in some cancers have been applied broadly to all metastatic diseases, despite evidence that these characteristics vary by tumor type. We will summarize how laminins influence the SCC metastatic process exclusively. Enhanced laminin protein deposition occurs at the invasive edge of SCC tumors, which correlates with elevated levels of laminin-binding β1 integrins on SCC cells, increased MMP-3 presence, worse prognosis, and lymphatic dissemination. Although these findings are significant, gaps in knowledge of the formation of a premetastatic niche, the processes of intra- and extravasation, and the contributions of the ECM to SCC metastatic cell dormancy persist. Bridging these gaps requires novel in vitro systems and animal models that reproduce tumor-stromal interactions and spontaneous metastasis seen in the clinic. These advances will allow accurate assessment of laminins to predict responders to transforming growth factor-β inhibitors and immunotherapy, as well as potential combinatorial therapies with the standard of care. Such clinical interventions may drastically improve quality of life and patient survival by explicitly targeting SCC metastasis.

Basement membrane defects in CD151-associated glomerular disease

BackgroundCD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3β1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease.MethodsNext-generation sequencing (NGS) was used to detect the variant in CD151. Electron and light microscopy were used to visualise the filtration barrier in the patient kidney biopsy, and immunoreactivity of patient red blood cells to anti-CD151/MER2 antibodies was performed. Further validation of the CD151 variant as disease-causing was performed in zebrafish using CRISPR-Cas9.ResultsWe report a young child with nail dystrophy and persistent urinary tract infections who was incidentally found to have nephrotic-range proteinuria. Through targeted NGS, a novel, homozygous truncating variant was identified in CD151, a gene rarely reported in patients with nephrotic syndrome. Electron microscopy imaging of patient kidney tissue showed thickening of GBM and podocyte effacement. Immunofluorescence of patient kidney tissue demonstrated that CD151 was significantly reduced, and we did not detect immunoreactivity to CD151/MER2 on patient red blood cells. CRISPR-Cas9 depletion of cd151 in zebrafish caused proteinuria, which was rescued by injection of wild-type CD151 mRNA, but not CD151 mRNA containing the variant sequence.ConclusionsOur results indicate that a novel variant in CD151 is associated with nephrotic-range proteinuria and microscopic haematuria and provides further evidence for a role of CD151 in glomerular disease. Our work highlights a functional testing pipeline for future analysis of patient genetic variants.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

Contribution of Endothelial Laminin-Binding Integrins to Cellular Processes Associated with Angiogenesis.

Endothelial cells engage extracellular matrix and basement membrane components through integrin-mediated adhesion to promote angiogenesis. Angiogenesis involves the sprouting of endothelial cells from pre-existing vessels, their migration into surrounding tissue, the upregulation of angiogenesis-associated genes, and the formation of new endothelial tubes. To determine whether the endothelial laminin-binding integrins, α6β4, and α3β1 contribute to these processes, we employed RNAi technology in organotypic angiogenesis assays, as well in migration assays, in vitro. The endothelial depletion of either α6β4 or α3β1 inhibited endothelial sprouting, indicating that these integrins have non-redundant roles in this process. Interestingly, these phenotypes were accompanied by overlapping and distinct changes in the expression of angiogenesis-associated genes. Lastly, depletion of α6β4, but not α3β1, inhibited migration. Taken together, these results suggest that laminin-binding integrins regulate processes associated with angiogenesis by distinct and overlapping mechanisms.

The laminin-binding integrins regulate nuclear factor κB-dependent epithelial cell polarity and inflammation.

The main laminin-binding integrins α3β1, α6β1 and α6β4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-κB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-κB-dependent signaling.

Tetraspanin 151 and its role in carcinogenesis

CD151, a member of the tetraspanin protein family, is a transmembrane protein involved in a number of physiological processes such as maintaining cell integrity, intercellular transport, wound healing, and more. It is not the main participant in these functions, but rather acts as a mediator molecule, interacting with other tetraspanins, integrins, adhesion receptors, signaling receptors and matrix metalloproteases. It is commonly expressed in endothelial cells and platelets, and its overexpression is associated with processes of progression and metastasis in neoplastic diseases. CD151, associated with laminin and laminin-binding integrins, is involved in the regulation of carcinogenesis. CD151 has been shown to play a significant role in the de novo carcinogenesis and progression of squamous cell carcinoma of the skin, influencing transcription factors related to the processes of cell proliferation and apoptosis. There are reports in the scientific literature of the association of impaired regulation of various tetraspanins with malignancies in humans. A number of authors have also reported that CD151 overexpression has been associated with a poor prognosis in cancers of the lung, colon, esophagus, pancreas and endometrium. In prostate cancer, overall survival rates were found to be significantly higher with low CD151 expression than with high expression. This tendency is particularly pronounced in highly and moderately differentiated tumors. Significant heterogeneity of CD151 expression in different tumor tissues was observed. The intensity of staining is noticeably lower in well-differentiated squamous cell carcinoma of the oral cavity. The expression gradient of CD151 is particularly noticeable at the front of tumor growth. Large-scale prospective and retrospective studies on various types of malignant tumors have established CD151 as a prognostic marker for tumor therapy with minimal side effects on normal cells.

Abstract LB256: Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia

Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) invasion, metastasis and decreased cancer-specific survival. Kindlins are integrin adaptors and mechanosensory focal adhesion (FA) proteins that activate and cluster integrins in response to structural ECM alterations in the tumor microenvironment. Hypoxia is a well-known inducer of tumor migration and invasion, results in ECM remodeling, and is physiologically relevant for PCa. The study's objective was to determine if integrin-kindlin-2 adhesion complexes were responsive to hypoxia and the dynamic distribution of integrin-kindlin-2 complexes using immunofluorescence microscopy (IFM). The methods included a combination of biochemical analysis of kindlin-2 adhesion complexes, qRT-PCR, immunoblotting, and immunocytochemistry. DU145 cells were analyzed in HEPES-containing media with 10% FBS under hypoxic conditions (1% O2) continuously exposed for 4, 8, 12, and 16 hours. The results showed that hypoxia increased the mRNA expression of CAIX, a well-known HIF-1α target gene, within 4 hours, resulting in 7-fold maximum expression. Similarly, VEGF-A mRNA was responsive to hypoxia as expected. In contrast, HIF-1α, α6 integrin, and kindlin-2 mRNA levels remained unchanged as compared to normoxia. Strikingly, we found that kindlin-2-containing adhesion complexes increased under hypoxia conditions as compared to normoxia and detected by immunoprecipitation, the complexes contained α6β1 integrin. It is important to note that the constitutive levels of either α6 integrin or kindlin-2 were not altered by hypoxia. Using IFM under normoxic conditions, we confirmed the location of kindlin-2 within phosphorylated (Y31) paxillin (pPXN)-containing FAs and within extended plasma membrane domains exclusive of pPXN. However, under hypoxic conditions, an increased reorganization of pPXN-containing kindlin-2 complexes occurred within 4 hours, was continuously changing up to 16 hours, with increasing fibrillar forms of FAs. In addition, kindlin-2 was observed as increased in plasma membrane protrusions devoid pPXN. The dynamic nature of the hypoxia-driven FAs was observed by a time course analysis and indicated the apparent assembly and disassembly of the structures during 16-hours of PCa cells exposed to hypoxia. We conclude that kindlin-2-integrin complexes are responsive to hypoxia and contain the unexpected α6β1 integrin in the membrane exclusive of FAs. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia [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 LB256.

Abstract LB233: Cancer cell invasion through a complex and dynamic smooth muscle microenvironment

Abstract The cancer-prone peripheral zone of the human prostate is a fibromuscular stroma bounded by a laminin-rich smooth muscle capsule. Aggressive tumors traverse the muscle to escape organ confinement, but the determinants of cell migration through this complex and highly elastic environment are not well understood. We modeled smooth muscle invasion using a unique in vivo model to test the role of mechanosensing, laminin-binding integrins (α6β1 and α3β1), collect cells successful in traversing smooth muscle, and identify transcriptomes involved in this complex invasive process. From the DU145 prostate cancer cell line (expressing integrins α3 and α6), we generated homozygous knockouts for genes ITGA6 (α6KO) and ITGA3 (α3KO). We tested for the ability to create 3D invasive cellular networks and measured biophysical features by electric cell-substrate impedance sensing. Muscle invasion was tested in vivo by injecting cells into the intraperitoneal cavity of male NSG mice. The cells colonized the inferior side of the respiratory diaphragm and invaded through to the superior surface after 6 weeks. We analyzed diaphragms by number of invasion sites and average invasion depth. A series of clones were also grown from wild-type, DU145 cells that had completely traversed the diaphragm muscle. Cells expressing integrins α6 and α3 produced 3D invasive networks on laminin-containing Matrigel and invaded into the diaphragm muscle in vivo. α6KO cells did not produce networks or invade muscle. Surprisingly, knockout of α3 increased 3D invasive networks, decreased cell-cell impedance, and altered in vivo muscle invasion. The results suggest that the integrins α6 and α3 are involved in the invasion of prostate cancer cells through a dynamic smooth muscle layer. The mechanosensing integrins are central to a variety of cell signaling programs. To further explore the transcriptional regulators of the muscle invasion process, we isolated RNA from cancer cells colonizing the inferior, superior surface and the intact diaphragm. Whole genome RNAseq revealed 1330 differentially expressed genes (DEGs) between inferior cells and cancer cells within the diaphragm, 788 DEGs between cells on the inferior and superior diaphragm surface, and 241 DEGs between cancer cells within the diaphragm and cells that reached the superior surface (padj <0.05, |fold change| >=1.3). Ingenuity Pathway Analysis revealed alterations in pathways including prostate cancer signaling, integrin signaling, other known cancer-related processes, and others. Through this in vivo assay we have the unique ability to parse out unexplored patterns of changes that occur in early muscle invasive disease to improve prostate cancer patient selection for curative treatment versus active surveillance. These studies were supported by NCI F30CA247106, P30CA23074, and R01CA24226. K.D.M is additionally supported by the ARCS Foundation Phoenix Chapter. Citation Format: Kendra D. Marr, Hina Arif-Tiwari, Jaime M. Gard, Noel E. Warfel, Raymond B. Nagle, Anne E. Cress. Cancer cell invasion through a complex and dynamic smooth muscle microenvironment [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 LB233.

Cell-Type-Specific Adhesiveness and Proliferation Propensity on Laminin Isoforms Enable Purification of iPSC-Derived Corneal Epithelium.

SummaryA treatment for intractable diseases is expected to be the replacement of damaged tissues with products from human induced pluripotent stem cells (hiPSCs). Target cell purification is a critical step for realizing hiPSC-based therapy. Here, we found that hiPSC-derived ocular cell types exhibited unique adhesion specificities and growth characteristics on distinct E8 fragments of laminin isoforms (LNE8s): hiPSC-derived corneal epithelial cells (iCECs) and other non-CECs rapidly adhered preferentially to LN332/411/511E8 and LN211E8, respectively, through differential expression of laminin-binding integrins. Furthermore, LN332E8 promoted epithelial cell proliferation but not that of the other eye-related cells, leading to non-CEC elimination by cell competition. Combining these features with magnetic sorting, highly pure iCEC sheets were fabricated. Thus, we established a simple method for isolating iCECs from various hiPSC-derived cells without using fluorescence-activated cell sorting. This study will facilitate efficient manufacture of iCEC sheets for corneal disease treatment and provide insights into target cell-specific scaffold selection.

CD151 in Respiratory Diseases.

The tetraspanin, Cluster of Differentiation 151 (CD151), is ubiquitously expressed in adult tissue, especially in the lungs where it has been implicated in lung cancer, asthma, influenza, and idiopathic pulmonary fibrosis (IPF). CD151 interacts with laminin-binding integrins and growth factor receptors, and is reported in cancer-promoting processes such as tumor initiation, metastasis, and angiogenesis. In asthma, CD151 was shown to promote airways hyperresponsiveness through calcium signaling whereas in influenza, CD151 was shown to be a novel host factor for nuclear viral export signaling. Furthermore, CD151 was shown to be associated with increased disease severity and poorer survival outcome in asthma and lung cancer, respectively. In this review, we provide an update on the current understanding of CD151 with regards to its contribution to lung pathophysiology. We also summarize factors that have been shown to regulate CD151 expression and identify key areas that need to be taken into consideration for its utility as a screening or prognostic tool in disease management and/or as a therapeutic target for the treatment of lung diseases.

Insights into Protein-Ligand Interactions in Integrin Complexes: Advances in Structure Determinations.

Integrins comprise a family of 24 heterodimeric transmembrane receptors that mediate cell attachment to the extracellular matrix and are critical for cell signaling. There are four classes of integrins: collagen-binding, Arg-Gly-Asp (RGD)-binding, laminin-binding, and leukocyte integrins. Owing to their involvement in modulating various critical cellular processes including proliferation, migration, differentiation, and survival, integrins have been investigated as therapeutic targets. Important progress has been made in the structural elucidation of integrins in recent years. Here we examine the protein-ligand interactions of integrin complexes and the related structure-based drug design of integrin inhibitors. Published integrin structures in the Protein Data Bank (PDB) are examined to gain insights into integrin-ligand interactions as well as the conformational dynamics of integrins.

Laminin is the ECM niche for trophoblast stem cells.

The niche is a specialized microenvironment for tissue stem cells in vivo. It has long been emphasized that niche ECM molecules act on tissue stem cells to regulate their behavior, but the molecular entities of these interactions remain to be fully elucidated. Here, we report that laminin forms the in vivo ECM niche for trophoblast stem cells (TSCs), the tissue stem cells of the placenta. TSCs expressed fibronectin-binding, vitronectin-binding, and laminin-binding integrins, whereas the integrin ligands present in the TSC niche were collagen and laminin. Therefore, the only niche integrin ligand available for TSCs in vivo was laminin. Laminin promoted TSC adhesion and proliferation in vitro in an integrin binding-dependent manner. Importantly, when the integrin-binding ability of laminin was genetically ablated in mice, the size of the TSC population was significantly reduced compared with that in control mice. The present findings underscore an ECM niche function of laminin to support tissue stem cell maintenance in vivo.

Laminin-binding integrins are essential for the maintenance of functional mammary secretory epithelium in lactation.

Integrin dimers α3/β1, α6/β1 and α6/β4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.