Interaction between Cell Adhesion Molecule Integrin α6 and Extracellular Matrix Laminin Promotes Myeloma Progression Via Akt Signaling

Background: Integrins are cell surface proteins and major cell adhesion transmembrane receptors that play an important role in cell adhesion andmultifaceted roles in cell growth, survival, and cytoskeleton formation by transmitting stimuli from ligands to the cell. Integrin α6 acts as a receptor for laminin, an extracellular matrix, and is involved in growth, invasion, and resistance to therapy in many cancers. However, the role of integrin α6 and interaction with laminin in multiple myeloma (MM) have not yet been investigated. Therefore, we focused on the regulation of integrin α6 expression in MM cells. Additionally, we focused on the binding of integrin α6 to laminin and examined proliferation and signal transduction by laminin binding.

Methods: ITGA6 expression was measured by RT-qPCR in CD138-positive plasma cells from 106 patients with MM, 13 patients with SMM, 72 patients with MGUS, 17 controls, and human myeloma cell lines (HMCL): KMS27, KMS11, KMS28BM, MM1S, KMS26, RPMI8226, and KMM1. The expression of integrin α6 on the HMCL cell surface was measured by flow cytometry. In HMCL, cell proliferation with laminin and molecules involved in signal transduction was evaluated using the CCK-8 assay and Western blotting.

Results: ITGA6 mRNA expression was significantly higher in the MM group than in the control and MGUS groups (p <0.001). The median ITGA6 mRNA expression levels in the control, MGUS, SMM, and MM groups were 0.359, 2.033, 35.230, and 7.961, respectively. There were no differences in ITGA6 expression in MM samples according to ISS (p=0.772) and cytogenetic risk (p=0.371). The overall survival and progression free survival did not differ between high and low ITGA6 expressions (OS; 5.1 years vs not reached; p = 0.394, PFS; 2.1 vs 2.2 years; p = 0.523).

To examine the regulation of integrin α6 expression by Myc, JQ1 and Myc inhibitors were added to the cells, and mean fluorescence intensity (MFI) decreased from 7.03 to 1.97, 6.15 to 2.54, and 5.71 to 2.31 in KMS27, KMS11, and KMS28BM, respectively, as JQ1 was added. MFI decreased from 4.23 to 3.08, 3.32 to 2.14, and 3.17 to 2.14 in KMS27, MM1S, and KMS28BM, respectively, as the Myc inhibitor was added.

Since it was reported that integrin α6 expression is regulated by HIF-1α, a hypoxia-inducible factor, we examined whether integrin α6 expression is regulated by HIF-1α in HMCL. Comparing cells cultured under normal oxygen and 1% O₂ conditions, the MFI of integrin α6 changed from 5.55 to 3.43, 1.43 to 1.51, 1.01 to 0.99, and 5.53 to 4.40 in KMS11, KMS26, RPMI8226, and KMM1, respectively.

We examined changes in cell proliferation caused by the binding of integrin α6 to laminin. We used laminins 111, 211, 411, and 511, which are known ligands for integrin α6. Laminin 411 promoted the proliferation of KMS27 and KMS11 cells expressing integrin α6 compared to those without laminin (p=0.0207, p=0.0532). In HMCL with high integrin α6 expression, stimulation with laminin 411 increased the phosphorylation of Akt and Erk1/2. KMS27 increased phosphorylated Akt by 12-fold, phosphorylated Erk1/2 by 2-fold, and 6-fold; KMS11 increased phosphorylated Akt by 15-fold, phosphorylated Erk1/2 by 34-fold, and 11-fold. Conversely, in RPMI8226 with no integrin α6 expression, stimulation of laminin 411 resulted in a 0.6- and 1-fold increase in phosphorylated Akt and Erk1/2, respectively.

Conclusion: RT-qPCR results suggest that integrin α6 may be involved in disease onset and progression. Cell surface expression of integrin α6 is regulated by Myc but not by HIF-1α. Stimulation of MM cells by laminin stimulated proliferation and caused phosphorylated Akt and Erk, suggesting that laminin is involved in MM proliferation via integrin α6. This study is a step toward developing integrin α6-targeted therapy.

Handa:Pfizer: Research Funding; Takeda Pharmaceuticals: Honoraria; Janssen Pharmaceutical: Honoraria, Research Funding; Amgen: Research Funding; GSK: Research Funding; Sanofi: Research Funding; AbbVie: Research Funding; Bristol Myers Squibb: Honoraria; Ono: Honoraria.