Integrins mediate bi-directional signaling between the extracellular ligand binding sites and the cytoplasmic tails, linking the cytoskeleton and intracellular processes to the cellular microenvironment. Interactions between the integrin cytoplasmic tail with accessory proteins can alter the integrins' ligand binding affinity, modulate cytoskeletal remodeling necessary for adhesion and motility and promote cell survival signaling. Prior studies have implicated involvement of α4 integrins in cell adhesion mediated drug resistance (CAM-DR) in cell models for multiple myeloma and AML. In the present study, we used a genetic reconstitution model to examine the requirement of α4-extracellular ligand interaction in CAM-DR and to delineate the α4 tail sequences mediating drug resistance.
JB4 cells, a Jurkat T-cell derivative lacking α4 expression, was reconstituted with wildtype α4 (JB4-α4) and a cytoplasmic tail truncated mutant (JB4-α4Δ). Cells were plated on CS1, Fn9.11 or BSA to specifically engage respectively, α4β1, α5β1, or no integrins. Adhered or non-adhered cells were untreated or treated with doxorubicin, and % apoptosis determined by flow cytometry using Annexin V binding.
The percentage of apoptotic Jurkat and JB4-α4 cells plated on CS1 or Fn9.11 and subjected to doxorubicin were half of those plated on BSA, indicating that both α4β1 and α5β1 integrin ligation confer enhanced chemoresistance. In contrast, JB4 cells lacking α4β1 expression exhibited CAM-DR when plated on Fn9.11, but not on CS1 or BSA, indicating α4β1-ligation to its substrate is necessary to support α4-mediated CAM-DR. Unexpectedly, JB4-α4Δ cells exhibited chemoresistance to doxorubicin when plated on all 3 substrates suggesting α4Δ expression resulted in an adhesion independent chemoresistant phenotype. α4Δ is truncated at the C-terminal tail following the highly conserved KxGFFKR sequence which is required for α-β heterodimer formation. This deletion is also known to disrupt α4Δβ1 binding with its extracellular ligand; a phenomenon which we confirmed using adhesion assays. The adhesion independent chemoresistance exhibited by JB4-α4Δ could be attributed to a gain in β1 expression (as α4Δβ1). To eliminate formation of the heterodimer, we created JB4-Tac and JB4-TacΔ cells, where TacΔ is a fusion of KxGFFKR to the extracellular and transmembrane epitope of the monomeric Tac. Treatment of JB4-TacΔ, but not JB4-Tac cells with doxorubicin in suspension recapitulated the low levels of apoptosis exhibited by JB4-α4Δ cells, indicating that the membrane proximal KxGFFKR sequence is sufficient to promote an adhesion independent form of chemoresistance.
The effects are mediated in part via stimulation of the PI3K/Akt/Bad cell survival pathway. Adhesion of α4β1-expressing cells to CS1, or of α5β1 to Fn9.11, stimulated an increase in phospho-Akt and phospho-Bad. Conversely, JB4-α4Δ and JB4-TacΔ cells exhibit constitutively high levels of phospho-Akt in an adhesion independent manner. We also show that the membrane proximal KxGFFKR associates with the Ca2+ binding protein calreticulin (CRT) that is known to regulate L-type Ca2+ channels. Finally, co-treatment of cells with doxorubicin and Verapamil, an L-type Ca2+ channel inhibitor, enhanced the chemosensitivity of JB4-TacΔ cells. Thus, the highly conserved membrane proximal KxGFFKR motif of α-integrins mediates chemoresistance via its interaction with a Ca2+ binding protein involved in regulation of an L-type calcium channel. With the increasing number of integrin-ligand interactions shown to promote CAM-DR, we speculate that interactions involving the KxGFFKR motif of α-integrins may be the common denominator for effecting cell adhesion mediated survival signaling and drug resistance.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.