Regulation of miRNA expression by α4ß1 integrin-dependent multiple myeloma cell adhesion.

The α4ß1 integrin regulates the trafficking of multiple myeloma (MM) cells and contributes to MM disease progression. MicroRNAs (miRNAs) can have both tumor suppressor and oncogenic roles and thus are key controllers of tumor evolution, and have been associated with different phases of MM pathogenesis. Using small RNAseq analysis, we show here that α4ß1-dependent MM cell adhesion regulates the expression of forty different miRNAs, therefore expanding our current view of the α4ß1 involvement in MM cell biology. Specific upregulation of miR-324-5p and miR-331-3p in cells attached to α4ß1 ligands was confirmed upon silencing the α4 integrin subunit, and their increased levels found to be dependent on Erk1/2- and PI3K-Akt-, but not Src-dependent signaling. Enhanced miR-324-5p expression upon α4ß1-mediated MM cell adhesion aimed the hedgehog (Hh) component SMO, revealing that the miR-324-5p-SMO module represents a α4ß1-regulated pathway that could control Hh-dependent cellular responses in myeloma. Our results open new therapy research avenues around the α4ß1 contribution to MM progression that deserve to be investigated.

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice.

ICAP-1 regulates ß1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4ß1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4ß1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers.

Upregulated expression and function of the α4ß1 integrin in multiple myeloma cells resistant to bortezomib.

The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes MM cell retention, survival, and resistance to different anti-MM agents, including proteasome inhibitors (PIs) such as bortezomib (BTZ). The α4ß1 integrin is a main adhesion receptor mediating MM cell-stroma interactions and MM cell survival, and its expression and function are downregulated by BTZ, leading to inhibition of cell adhesion-mediated drug resistance (CAM-DR) and MM cell apoptosis. Whether decreased α4ß1 expression and activity are maintained or recovered upon development of resistance to BTZ represents an important question, as a potential rescue of α4ß1 function could boost MM cell survival and disease progression. Using BTZ-resistant MM cells, we found that they not only rescue their α4ß1 expression, but its levels were higher than in parental cells. Increased α4ß1 expression in resistant cells correlated with enhanced α4ß1-mediated cell lodging in the BM, and with disease progression. BTZ-resistant MM cells displayed enhanced NF-κB pathway activation relative to parental counterparts, which contributed to upregulated α4 expression and to α4ß1-dependent MM cell adhesion. These data emphasize the upregulation of α4ß1 expression and function as a key event during resistance to BTZ in MM, which might indirectly contribute to stabilize this resistance, as stronger MM cell attachment to BM stroma will regain CAM-DR and MM cell growth and survival. Finally, we found a strong correlation between high ITGB1 (integrin ß1) expression in MM and poor progression-free survival (PFS) and overall survival (OS) during treatment of MM patients with BTZ and IMIDs, and combination of high ITGB1 levels and presence of the high-risk genetic factor amp1q causes low PFS and OS. These results unravel a novel prognostic value for ITGB1 in myeloma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The good and bad faces of the CXCR4 chemokine receptor.

Chemokines are chemotactic cytokines that promote cell migration and activation under homeostatic and inflammatory conditions. Chemokines bind to seven transmembrane-spanning receptors that are coupled to heterotrimeric guanine nucleotide-binding (G) proteins, which are the responsible for intracellularly transmitting the activating signals for cell migration. Hematopoiesis, vascular development, lymphoid organ morphogenesis, cardiogenesis and neural differentiation are amongst the processes involving chemokine function. In addition, immune cell trafficking from bone marrow to blood circulation, and from blood and lymph to lymphoid and inflamed tissues, is tightly regulated by chemokines both under physiological conditions and also in autoimmune diseases. Furthermore, chemokine binding to their receptors stimulate trafficking to and positioning of cancer cells into target tissues and organs during tumour dissemination. The CXCL12 chemokine (also known as stromal-cell derived factor-1α, SDF-1α) plays key roles in hematopoiesis and lymphoid tissue architecture, in cardiogenesis, vascular formation and neurogenesis, as well as in the trafficking of solid and hematological cancer cell types. CXCL12 binds to the CXCR4 receptor, a multi-facetted molecule which tightly mirrors CXCL12 functions in homeostasis and disease. This review addresses the important roles of the CXCR4-CXCL12 axis in homeostasis, specially focusing in hematopoiesis, as well as it provides a picture of CXCR4 as mediator of cancer cell spreading, and a view of the available CXCR4 antagonists in different cancer types.

Lateral Mobility and Nanoscale Spatial Arrangement of Chemokine-activated α4ß1 Integrins on T Cells.

Chemokine stimulation of integrin α4ß1-dependent T lymphocyte adhesion is a key step during lymphocyte trafficking. A central question regarding α4ß1 function is how its lateral mobility and organization influence its affinity and avidity following cell stimulation with chemokines and/or ligands. Using single particle tracking and superresolution imaging approaches, we explored the lateral mobility and spatial arrangement of individual α4ß1integrins on T cells exposed to different activating stimuli. We show that CXCL12 stimulation leads to rapid and transient α4ß1activation, measured by induction of the activation epitope recognized by the HUTS-21 anti-ß1antibody and by increased talin-ß1 association. CXCL12-dependent α4ß1 activation directly correlated with restricted lateral diffusion and integrin immobilization. Moreover, co-stimulation by CXCL12 together with soluble VCAM-1 potentiated integrin immobilization with a 5-fold increase in immobile integrins compared with unstimulated conditions. Our data indicate that docking by talin of the chemokine-activated α4ß1 to the actin cytoskeleton favors integrin immobilization, which likely facilitates ligand interaction and increased adhesiveness. Superresolution imaging showed that the nanoscale organization of high-affinity α4ß1 remains unaffected following chemokine and/or ligand addition. Instead, newly activated α4ß1 integrins organize on the cell membrane as independent units without joining pre-established integrin sites to contribute to cluster formation. Altogether, our results provide a rationale to understand how the spatiotemporal organization of activated α4ß1 integrins regulates T lymphocyte adhesion.

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4ß1.

Stimulation by chemokines of integrin α4ß1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4ß1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4ß1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4ß1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4ß1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4ß1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4ß1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4ß1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.