Calpain-dependent clearance of the autophagy protein p62/SQSTM1 is a contributor to ΔPK oncolytic activity in melanoma

Abstract

Oncolytic virotherapy is a promising strategy for reducing tumor burden through selective virus replication in rapidly proliferating cells. However, the lysis of slowly replicating cancer stem cells (CSCs), which maintain neoplastic clonality, is relatively modest and the potential contribution of programmed cell death pathways to oncolytic activity is still poorly understood. We show that the oncolytic virus ΔPK lyses CSC-enriched breast cancer and melanoma 3D spheroid cultures at low titers (0.1 pfu/cell) without resistance development and it inhibits the 3D growth potential (spheroids and agarose colonies) of melanoma and breast cancer cells. ΔPK induces calpain activation in both melanoma and breast cancer 3D cultures as determined by the loss of the p28 regulatory subunit, and 3D growth is restored by treatment with the calpain inhibitor PD150606. In melanoma, ΔPK infection also induces light chain 3 (LC3)-II accumulation and p62/SQSTM1 clearance, both markers of autophagy, and 3D growth is restored by treatment with the autophagy inhibitor chloroquine (CQ). However, expression of the autophagy-required protein Atg5 is not altered and CQ does not restore p62/SQSTM1 expression, suggesting that the CQ effect may be autophagy-independent. PD150606 restores expression of p62/SQSTM1 in ΔPK-infected melanoma cultures, suggesting that calpain activation induces anti-tumor activity through p62/SQSTM1 clearance.

Figure 1. ΔPK has oncolytic activity in breast cancer and melanoma spheroid cultures

(a) HS578T spheroid cultures were mock- or ΔPK-infected (moi=1) and examined at 48 hrs p.i. (20×). Similar results were obtained for A2058 and A375 cells. (b) Mock- or ΔPK-infected A2058 spheroid cultures were dissociated with accutase stained with PI and analyzed by FCM. Similar results were obtained for A375 and HS578T cells. (c) HS578T and A2058 cells mock infected or infected with ΔPK (moi=1; 48 hrs) were assayed for growth in spheroid culture and the results are expressed as No. of spheroids/1×104 cells ± SD. Similar results were obtained for A375 cells and for colony forming potential in soft agar.

Figure 2. Low titers of ΔPK penetrate and lyse 3-D spheroids without resistance development

(a) A2058 spheroid cultures were infected with ΔPK at moi= 10, 1 or 0.1, virus was removed by centrifugation and the spheroids were re-plated in fresh, virus-free medium and counted as described in Materials and methods over a period of 10 days p.i. Spheroid counts decreased with time p.i. and were no longer seen by day 10 p.i. (arrow) (b) Spheroids mock-infected or infected with ΔPK at moi=10 and 0.1 were stained with PI at the listed times p.i. and imaged under phase contrast (top panels) and red fluorescence microscopy (bottom panels). Representative images are shown and similar results were obtained for A375 and HS578T cells. Arrow indicates a remaining viable cell.

Figure 3. Lysis of spheroid cultures independent of virus replication

(a) HS578T and A2058 spheroid cultures were infected with ΔPK (moi=1; 48hrs) or mock-478 infected with PBS and stained with PI or double stained with PI and antibodies to the CSC phenotypic markers CD271 (A2058) or CD44/CD24 (HS578T) and examined by FCM using gates drawn based on forward and side scatter and isotype control staining patterns. Results are expressed as % PI+ cells calculated relative to the total number of cells in the spheroid cultures (total) or the CD44⁺/CD24^low/- or CD271⁺ gated cells ± SD. (b,c). Dissociated spheroid cultures mock or ΔPK infected as in (a) were co-stained with antibodies to VP5 and the respective CSC markers CD44/CD24 (b) or CD271 (c) and the % staining cells counted as described in Materials and Methods. Results are expressed as % VP5+ cells calculated relative to the total number of cells in the spheroid cultures (total) or the % marker+ cells ± SEM. (d) A2058, HS578T and WI-38 cells were infected with ΔPK (moi = 1), examined for virus growth in serum-free medium and virus titers were determined by plaque assay. Results are expressed as mean pfu/cell (burst size). ΔPK is growth restricted in A2058 and HS578T cells and does not grow in WI-38 cells.

Figure 4. Cell type-specific death pathways contribute to ΔPK oncolytic activity

(a-c). A2058, A375 and HS578T 2-D cultures were mock-infected or infected with ΔPK (moi = 1; 48hrs) in the absence or presence of PD150606 (100μM), CQ (10μM) or zVAD-fmk (20μM) and plated under soft agar as described in Materials and methods. Colonies were counted and the results are expressed as % colonies ± SD calculated relative to the mock-infected cultures (100%). Similar results were obtained for growth in spheroid cultures. (d). A2058 cultures infected with ΔPK in the absence or presence of the inhibitors were assayed for virus growth by plaque assay at 4-36 hrs p.i. and the results are expressed as pfu/cell ± SD. Similar results were obtained for A375 and HS578T cells.

Figure 5. ΔPK induces LC3-II accumulation and calpain-dependent clearance of p62/SQSTM1

(a) A2058 and A375 spheroid cultures were dissociated into single cell suspensions and mock- or ΔPK-infected (moi=1) for 1,4 or 24 hrs and protein extracts were immunoblotted with antibodies to LC3 followed by actin (loading control). Data were quantified by densitometric scanning and the results obtained for three replicate experiments are expressed as LC3-II/LC3-I ratio. (b) A2058 spheroid cultures dissociated into single cell suspensions were mock- or ΔPK-infected (moi=1; 24hrs) in the absence or presence of PD150606 (100 μM) or CQ (10μM) and protein extracts were immunoblotted with antibodies to Atg5, p62/SQSTM1, the calpain p28 regulatory subunit, AIF, or GAPDH (loading control). The blots were stripped between antibodies and representatives of three replicate experiments are shown. Similar results were obtained in A375 spheroids. Data were quantified by densitometric scanning and the results obtained for three replicate experiments are expressed as densitometric units ± SD for Atg5, p62/SQSTM1 and p28 (calpain).