Matrix stiffness drives stromal autophagy and promotes formation of a protumorigenic niche

Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV–focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.

Adenine Inhibits the Invasive Potential of DLD-1 Human Colorectal Cancer Cell via the AMPK/FAK Axis

Tumor metastasis is a major cause of death of patients with colorectal cancer (CRC). Our previous findings show that adenine has antiproliferation activity against tumor cells. However, whether adenine reduces the invasiveness of DLD-1 and SW480 CRC cells has not been thoroughly explored. In this study, we aimed to explore the effects of adenine on the invasion potential of DLD-1 cells. Our findings showed that adenine at concentrations of ≤200 μM did not influence the cell viability of DLD-1 and SW480 CRC cells. By contrast, adenine reduced the migratory potential of the CRC cells. Moreover, it decreased the invasion capacity of the CRC cells in a dose-dependent manner. We further observed that adenine downregulated the protein levels of tissue plasminogen activator, matrix metalloproteinase-9, Snail, TWIST, and vimentin, but upregulated the tissue inhibitor of metalloproteinase-1 expression in DLD-1 cells. Adenine decreased the integrin αV level and reduced the activation of integrin-associated signaling components, including focal adhesion kinase (FAK), paxillin, and Src in DLD-1 cells. Further observations showed that adenine induced AMP-activated protein kinase (AMPK) activation and inhibited mTOR phosphorylation in DLD-1 cells. The knockdown of AMPK restored the reduced integrin αV level and FAK/paxillin/Src signaling inhibited by adenine in DLD-1 cells. Collectively, these findings reveal that adenine reduces the invasion potential of DLD-1 cells through the AMPK/integrin/FAK axis, suggesting that adenine may have anti-metastatic potential in CRC cells.

Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.

Mechanotransduction via the coordinated actions of integrins, PI3K signaling and Connexin hemichannels

Mechanical loading opens connexin 43 (Cx43) hemichannels (HCs), leading to the release of bone anabolic molecules, such as prostaglandins, from mechanosensitive osteocytes, which is essential for bone formation and remodeling. However, the mechanotransduction mechanism that activates HCs remains elusive. Here, we report a unique pathway by which mechanical signals are effectively transferred between integrin molecules located in different regions of the cell, resulting in HC activation. Both integrin α5 and αV were activated upon mechanical stimulation via either fluid dropping or flow shear stress (FSS). Inhibition of integrin αV activation or ablation of integrin α5 prevented HC opening on the cell body when dendrites were mechanically stimulated, suggesting mechanical transmission from the dendritic integrin αV to α5 in the cell body during HC activation. In addition, HC function was compromised in vivo, as determined by utilizing an antibody blocking αV activation and α5-deficient osteocyte-specific knockout mice. Furthermore, inhibition of integrin αV activation, but not that of α5, attenuated activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway upon mechanical loading, and the inhibition of PI3K/AKT activation blocked integrin α5 activation and HC opening. Moreover, HC opening was blocked only by an anti-integrin αV antibody at low but not high FSS levels, suggesting that dendritic αV is a more sensitive mechanosensor than α5 for activating HCs. Together, these results reveal a new molecular mechanism of mechanotransduction involving the coordinated actions of integrins and PI3K/AKT in osteocytic dendritic processes and cell bodies that leads to HC opening and the release of key bone anabolic factors.

Hippocampal and Prefrontal Cortical Brain Tissue Levels of Irisin and GDF15 Receptor Subunits in Children

Cold-stress hormones (CSHs) stimulate thermogenesis and have direct neuroprotective effects on the brain. The obligatory receptor components of two new CSHs (irisin and growth differentiation factor-15 [GDF15]) were recently discovered. Irisin binds integrin-αV/β5 heterodimers while GDF-15 binds to the orphan receptor glial cell-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL). In addition, integrin-αV/β5 was just identified as the key receptor mediating Zika virus infection in the CNS. We measured integrin-αV, integrin-β5, and GFRAL protein levels across 78 high-quality human male/female brain tissues in infants, toddlers, preschoolers, adolescent, and adults—providing the most robust analysis to date on their levels in the human cortex and hippocampus. We report that integrin-αV was detected at all ages in the prefrontal cortex with levels greatest in adults. Integrin-αV was also detected in the hippocampus in all age groups. In contrast, integrin-β5 was detected in cortex and hippocampus largely restricted to infants. Co-expression of integrin-αV/β5 in the human infant hippocampus and cortex suggests the possibility that irisin has a more robust effect on the developing vs. the adult brain and may have implications for Zika virus infection in infants and young children.

HSPA1L Enhances Cancer Stem Cell-Like Properties by Activating IGF1Rβ and Regulating β-Catenin Transcription

Studies have shown that cancer stem cells (CSCs) are involved in resistance and metastasis of cancer; thus, therapies targeting CSCs have been proposed. Here, we report that heat shock 70-kDa protein 1-like (HSPA1L) is partly involved in enhancing epithelial–mesenchymal transition (EMT) and CSC-like properties in non-small cell lung cancer (NSCLC) cells. Aldehyde dehydrogenase 1 (ALDH1) is considered a CSC marker in some lung cancers. Here, we analyzed transcriptional changes in genes between ALDH1high and ALDH1low cells sorted from A549 NSCLC cells and found that HSPA1L was highly expressed in ALDH1high cells. HSPA1L played two important roles in enhancing CSC-like properties. First, HSPA1L interacts directly with IGF1Rβ and integrin αV to form a triple complex that is involved in IGF1Rβ activation. HSPA1L/integrin αV complex-associated IGF1Rβ activation intensified the EMT-associated cancer stemness and γ-radiation resistance through its downstream AKT/NF-κB or AKT/GSK3β/β-catenin activation pathway. Secondly, HSPA1L was also present in the nucleus and could bind directly to the promoter region of β-catenin to function as a transcription activator of β-catenin, an important signaling protein characterizing CSCs by regulating ALDH1 expression. HSPA1L may be a novel potential target for cancer treatment because it both enhances IGF1Rβ activation and regulates γβ-catenin transcription, accumulating CSC-like properties.

Exploring Single Nucleotide Polymorphisms in ITGAV for Gastric, Pancreatic and Liver Malignancies: An Approach towards Biomarker Discovery

Background: Integrin αV, encoded by ITGAV gene is one of the most studied protein subunits, closely associated with liver, pancreatic and stomach cancer progression and metastasis via regulation of angiogenesis. Occurrence of Single Nucleotide Polymorphisms (SNPs) in cancer-associated proteins is a key determinant for varied susceptibility of an individual towards cancer. Methodology: The study investigated the deleterious effects of these cancer-associated SNPs on the protein’s structure, stability and cancer causing potential using an in silico approach. Numerous computational tools were employed that identified the most deleterious cancer-associated SNPs and those to get actively involved in post-translational modifications. Impact of these SNPs on the protein structure, function and stability was also examined. Conclusion and Future Scope: A total 63 non-synonymous SNPs in ITGAV gene were observed to be associated in these three gastrointestinal cancers and among this 63, 19 were the most deleterious ones. The structural and functional importance of residues altered by most damaging SNPs was analyzed through evolutionary conservation and solvent accessibility. The study also elucidated three-dimensional structures of the 19 most damaging mutants. The analysis of conformational variation identified 5SNPs (D379Y, G188E, G513V, L950P, and R540L) in integrin αV, which influence protein’s structure. Three calcium binding sites were predicted at residues: D379, G384 and G408 and a peptide binding site at residue: R369 in integrin αV. Therefore, SNPs D379Y, G384C, G408R and R369W have the potential to alter the binding properties of the protein. Screening and characterization of deleterious SNPs could advance novel biomarker discovery and therapeutic development in future.