Sensitivity and Resistance of Uveal Melanoma (UM) cells to PKC inhibition: Role of the Steroid Receptor Coactivator (SRC)-3

Abstract

 

Purpose

 

We have previously reported that mutant G proteins (Gαq and Gα11) in UM promote, via the protein kinase C (PKC) pathway, the post-translational stabilization of SRC-3, a multifunctional transcriptional coactivator that is frequently overexpressed and confers poor prognosis in many types of cancers, including melanoma. G protein-mutant UM cells are exquisitely dependent on PKC activity and SRC-3 expression for their survival in vitro. We now examined the role of SRC-3 as a therapeutic target and as a downstream mediator of the PKC pathway in animal models of UM.

 

Methods

 

We transfected GNAQ-mutant Mel202 cells with a plasmid expressing SRC-3 shRNA (2 different sequences) or control vector, under a tetracycline-inducible promoter. Cells were injected subcutaneously (sc) into immunocompromized mice and treated with doxycycline (provided in the drinking water). We also treated a xenograft model of 92.1 GNAQ-mutant UM cells with the PKC small molecule inhibitor (SMI) PKC412 (midostaurin).

 

Results

 

Doxycycline-induced SRC-3 shRNA suppressed SRC-3 expression and exerted potent anticancer activity against UM cells in vitro and in vivo. The PKC SMI PKC412 suppressed SRC-3 protein expression in our UM xenograft model (92.1 cells) and exerted potent anticancer activity in vivo, prolonging median survival (not reached yet after 8 months of PKC412 treatment) compared to the vehicle-treated cohort (2 weeks, see Fig. A). Acute restoration of SRC-3 expression by adenovirus rescued UM cells from the anticancer effect of PKC412. UM (92.1) cells with acquired resistance to PKC412 that emerged after prolonged treatment in vivo were found to express restored SRC-3 protein levels.

 

Conclusions

 

SRC-3 is an important therapeutic target in UM in vitro and in vivo. We propose that SRC-3 is a downstream mediator of PKC signaling and that SRC-3 inhibition could restore sensitivity to PKC SMIs.

 

 

Treatment of 92.1 UM cells xenografted sc into immunocompromized mice with PKC412 (100mg/kg/day, 5 days/week) prolonged median survival (not reached yet after 8 months of treatment) compared to vehicle-treated controls (2 weeks, A). IHC revealed loss of SRC-3 expression in UM xenografts from PKC412-treated mice (C) compared to the vehicle-treated mice (B). In the PKC412-treated mice that have died so far, IHC revealed that the PKC412-resistant UM xenografts express significantly higher levels of SRC-3 (D).

 

Keywords: 589 melanoma • 714 signal transduction • 624 oncology