L-Type Cav 1.2 Calcium Channel-α-1C Regulates Response to Rituximab in Diffuse Large B-Cell Lymphoma

Abstract

Purpose: One third of patients with diffuse large B-cell lymphoma (DLBCL) succumb to the disease partly due to rituximab resistance. Rituximab-induced calcium flux is an important inducer of apoptotic cell death, and we investigated the potential role of calcium channels in rituximab resistance.

Experimental design: The distinctive expression of calcium channel members was compared between patients sensitive and resistant to rituximab, cyclophosphamide, vincristine, doxorubicin, prednisone (RCHOP) regimen. The observation was further validated through mechanistic in vitro and in vivo studies using cell lines and patient-derived xenograft mouse models.

Results: A significant inverse correlation was observed between CACNA1C expression and RCHOP resistance in two independent DLBCL cohorts, and CACNA1C expression was an independent prognostic factor for RCHOP resistance after adjusting for International Prognostic Index, cell-of-origin classification, and MYC/BCL2 double expression. Loss of CACNA1C expression reduced rituximab-induced apoptosis and tumor shrinkage. We further demonstrated direct interaction of CACNA1C with CD20 and its role in CD20 stabilization. Functional modulators of L-type calcium channel showed expected alteration in rituximab-induced apoptosis and tumor suppression. Furthermore, we demonstrated that CACNA1C expression was directly regulated by miR-363 whose high expression is associated with worse prognosis in DLBCL.

Conclusions: We identified the role of CACNA1C in rituximab resistance, and modulating its expression or activity may alter rituximab sensitivity in DLBCL.

Figure 1:. CACNA1C expression and significance in DLBCL patients and normal B cells.

(A) The mRNA expression of CACNA1C was lower in rituximab-resistant patients compared to rituxixmab-sensitive patients (p=0.009), while CD20 mRNA expression showed no correlation (p=NS). (B) The protein expression of CACNA1C was negative in one case with rituximab resistance and positive in the other case with rituximab sensitivity, while CD20 protein expression was positive in both of them. (C) In an independent DLBCL cohort, there was no significant difference in CACNA1C mRNA expression between GCB-DLBCL and ABC-DLCBL subtypes (p=NS). (D) Of the normal B-cell subsets, centrocytes and plasmablast showed comparatively lower expression of CACNA1C. (E) The patients with lowest quartile of CACNA1C expression showed inferiors OS (HR=1.974, p=0.020).

Figure 2:. The effect of CACNA1C downregulation on rituximab-induced apoptosis and tumor inhibition.

(A and B) The interference with shRNA to CACNA1C reduced the percent of rituximab-induced cell apoptosis (p<0.05) in OCI-ly7 and OCI-Ly8 cell line. (C) In xenograft mice models of OCI-ly7 cell line, downregulation of CACNA1C by its shRNA obviously suppressed rituximab-induced tumor shrinkage in tumor volume and weight (p<0.05).

Figure 3:. The interaction between CACNA1C and CD20 molecules during rituximab action.

(A) CD20 (green light) and CACNA1C (red light) molecules exhibited a similar polarized distribution after rituximab treatment of 0, 2, and 12 h. Their overlapping distribution (yellow light) was captured in the plasma membrane in the merged phase. (B) CACNA1C protein was detected in anti-CD20 pull-down protein using western blot after rituximab treatment of 2 h in OCI-ly7 cells. (C) Knockdown of CACNA1C expression with transduced shRNAs did not affect CD20 mRNA levels, but downregulated CD20 protein expression. Other isoforms of α1 subunits, such as CACNA1D, CACNA1F, and CACNA1S, showed no obvious alteration. (D) Proteasome inhibitor bortezomib (20nM) restored CD20 expression after administration of 6 h in OCI-ly7 cells.

Figure 4:. The influence of L-type calcium channel modulators on rituximab-induced apoptosis and tumor suppression.

(A) The agonist of L-type calcium (Bay K8644) enhanced the rituximab-induced apoptosis, while the antagonist of L-type calcium channel (nimodipine) attenuated the rituximab-induced apoptosis (p<0.01) in DLBCL cell lines. (B) In PDX mouse models, rituximab combined with agonist (Bay K8644) markedly enhanced rituximab action (p<0.05). Neither its combination with antagonist (nimodipine) nor modulators of L-type calcium channel alone showed obvious effect on tumor growth (p>0.05).

Figure 5:. The association of miRNA-363 with CACNA1C expression and rituximab-induced apoptosis.

(A) The miRNA-363 inhibited the luciferase activity in 293T cells with wild-type (WT) 3’UTR of CACNA1C gene, but did not affect it in cells with mutant 3’UTR of CACNA1C gene (p<0.01). (B) The ectopic expression of miRNA-363 reduced CACNA1C and CD20 protein expression in OCI-ly7 and OCI-ly8 cell lines. (C) The ectopic expression of miRNA-363 decreased rituximab-induced apoptosis in OCI-ly7 and OCI-ly8 cell lines (p<0.05). (D) The knockout of miRNA-363 led to the upregualtion of CACNA1C expression and (E) the increase of rituximab-induced apoptosis (p<0.05).