Survival of human multiple myeloma cells is dependent on MUC1 C-terminal transmembrane subunit oncoprotein function

Abstract

The MUC1 C-terminal transmembrane subunit (MUC1-C) oncoprotein is a direct activator of the canonical nuclear factor-kappaB (NF-kappaB) RelA/p65 pathway and is aberrantly expressed in human multiple myeloma cells. However, it is not known whether multiple myeloma cells are sensitive to the disruption of MUC1-C function for survival. The present studies demonstrate that peptide inhibitors of MUC1-C oligomerization block growth of human multiple myeloma cells in vitro. Inhibition of MUC1-C function also blocked the interaction between MUC1-C and NF-kappaB p65 and activation of the NF-kappaB pathway. In addition, inhibition of MUC1-C in multiple myeloma cells was associated with activation of the intrinsic apoptotic pathway and induction of late apoptosis/necrosis. Primary multiple myeloma cells, but not normal B-cells, were also sensitive to MUC1-C inhibition. Significantly, treatment of established U266 multiple myeloma xenografts growing in nude mice with a lead candidate MUC1-C inhibitor resulted in complete tumor regression and lack of recurrence. These findings indicate that multiple myeloma cells are dependent on intact MUC1-C function for constitutive activation of the canonical NF-kappaB pathway and for their growth and survival.

Fig. 1.

GO-201 induces the arrest of U266 and RPMI8226 cell growth. A, amino acid sequences of GO-201 and CP-1 linked to a poly(arginine) transduction domain (Raina et al., 2009). U266 cells were left untreated (♦) and treated with 5 μM GO-201 (▲) or CP-1 (■) each day for 3 days. Viable cell number was determined by trypan blue exclusion. B, U266 cells were left untreated (Control) and treated with 5 μM GO-201 or CP-1 each day for 3 days. Cells were fixed and analyzed for cell cycle distribution by flow cytometry. C and D, RPMI8226 cells were left untreated (CTL) and treated with 5 μM GO-201 or CP-1 each day for 3 days. Viable cell number as determined by trypan blue exclusion is expressed as the mean ± S.D. of three determinations (C). Cells were analyzed for cell cycle distribution (D).

Fig. 2.

GO-201 blocks nuclear targeting of NF-κB p65 and induces late apoptosis/necrosis. A and B, U266 (A) and RPMI8226 (B) cells were left untreated (Control) and treated with 5 μM GO-201 or CP-1 each day for 2 days. Nuclear lysates were immunoblotted with the indicated antibodies. C and D, U266 (C) and RPMI8226 (D) cells were left untreated (CTL) and treated with 5 μM GO-201 or CP-1 each day for 3 days. Cells were stained with PI/annexin V and analyzed by flow cytometry (left). The percentage of cells positive for both PI and annexin V is indicated in the top right quadrants. The results are expressed as the percentage (mean ± S.D. of three determinations) of late apoptotic/necrotic cells (right).

Fig. 3.

GO-203 is an effective inhibitor of multiple myeloma cell growth and survival. A, d-amino acid sequences of GO-203 and CP-2 linked to a poly(arginine) transduction domain. U266 cells were left untreated (CTL) and treated with 5 μM GO-203 or CP-2 each day for 4 days. Viable cell number as determined by trypan blue exclusion is expressed as the mean ± S.D. of three determinations. B, RPMI8226 cells were left untreated (♦) and treated with 5 μM GO-203 (▲) or CP-2 (■) each day for the indicated days. Viable cell number was determined by trypan blue exclusion. C, primary multiple myeloma cells were isolated from the bone marrow of a patient with >90% CD138+ cells, incubated with a control IgG or an anti-MUC1 antibody and analyzed by flow cytometry (top). The percentage of MUC1-positive cells is indicated. The primary multiple myeloma cells were left untreated (CTL) and treated with 5 μM GO-203 or CP-2 each day for 6 days. Viable cell number as assessed by trypan blue exclusion is expressed as the mean ± S.D. of three determinations (bottom). P values were determined by the Student's t test. D, normal peripheral blood B-cells were left untreated (♦) and treated with 5 μM (■) or 10 μM (▲) GO-203 each day for the indicated days. Viable cell number was determined by trypan blue exclusion.

Fig. 4.

GO-203 inhibits constitutive activation of the NF-κB p65 pathway. U266 (left) and RPMI8226 (right) cells were left untreated (Control) and treated with 5 μM GO-203 or CP-2 each day for 2 days. A, anti-MUC1-C precipitates were immunoblotted with anti-p65 and anti-MUC1-C. B, nuclear lysates were immunoblotted with the indicated antibodies. C, soluble chromatin was precipitated with anti-p65. The final DNA extractions were amplified by polymerase chain reaction with pairs of primers that cover the NF-κB response element (−597 to −304) in the Bcl-xL promoter. D, Whole-cell lysates were immunoblotted with the indicated antibodies.

Fig. 5.

GO-203 induces activation of the intrinsic apoptotic pathway. A, U266 cells were left untreated (Control) and treated with 5 μM GO-203 or 5 μM CP-2 each day for 3 days. For the GO-203-treated cells, 5 μM zVAD-fmk was added during the last 24 h. Cells were stained with PI/annexin V and analyzed by flow cytometry. The percentage of cells positive for both PI and annexin V is indicated in the top right quadrants (left). The results are expressed as the percentage (mean ± S.D. of three determinations) of late apoptotic/necrotic cells (right). B, U266 cells were left untreated (CTL) and treated with 5 μM GO-203 or 5 μM CP-2 each day for 3 days. ROS levels were determined by oxidation of HE and flow cytometry (left). The results are expressed as relative ROS levels (mean ± S.D. of three determinations) compared with that obtained with control cells (right). C and D, U266 (C) and RPMI8226 (D) cells were left untreated (Control) and treated with 5 μM GO-203 or CP-2 each day for 3 days. Lysates were immunoblotted with the indicated antibodies.

Fig. 6.

GO-203 induces complete regressions of U266 tumors. BALB/c nu/nu mice were injected subcutaneously in the flank with 10⁷ U266 cells. The mice were pair-matched when the tumors were ∼100 mm³. Treatment groups consisted of 10 mice injected intraperitoneally with PBS (vehicle control; ■), 30 mg/kg GO-203 (●), or 30 mg/kg CP-2 (▲) each day for 21 days. Another group was treated with 30 mg/kg GO-203 administered each day for 5 days per week for 3 weeks (♦). Mice were weighed twice weekly, and tumor measurements were performed every 2 days. There was no weight loss in any of the groups. The results are expressed as the mean tumor volume with a S.E. of <15% (A). There was no evidence for recurrence in the two GO-203 treatment groups at 180 days. U266 tumors and tumor implantation sites harvested on day 28 were stained with H&E (B).