New data, published in Cancer Research, show that androgen receptor (AR) expression and phosphorylation are increased in sunitinib-resistant kidney cancer. Combination treatment with the AR antagonist enzalutamide restored sensitivity to sunitinib in experimental models, providing a rationale for clinically testing this combination in sunitinib-resistant disease.

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Patrick Morgan/Macmillan Publishers Limited

Integrative analysis of a reverse-phase protein array of a patient-derived xenograft (PDX) model of acquired sunitinib resistance showed increased AR expression. In vitro, kidney cancer cell lines with either acquired or intrinsic sunitinib resistance also showed increased AR expression. AR levels correlated with sunitinib sensitivity in AR-positive and AR-negative cell lines. Overexpression of wild-type AR in AR-negative cells decreased sensitivity to sunitinib. Combined treatment of sunitinib and enzalutamide partially restored sunitinib sensitivity.

Combination treatment with sunitinib and enzalutamide had a synergistic effect in sunitinib-resistant, AR-positive cells, but enzalutamide alone did not significantly affect cell viability. Sunitinib treatment in AR-positive cells induced expression of AR target genes, including KLK2, KLK4, ZBTB16, and MYC, which co-treatment with enzalutamide inhibited. In culture supernatants, levels of KLK2 were increased in sunitinib-treated, sunitinib-resistant cells, but combination treatment with enzalutamide reduced these levels.

Combination treatment with sunitinib and enzalutamide had a synergistic effect in sunitinib-resistant, AR-positive cells

The absence of androgens in the culture media did not affect AR expression or cell growth. Mass spectrometry of PDX models with acquired or intrinsic sunitinib resistance and kidney cancer cell lines did not show presence of considerable levels of androgens. Immunofluorescence showed increased AR nuclear translocation and phosphorylation of serine 81 in AR-positive cells after sunitinib treatment. Cyclin-dependent kinase 1 (CDK1), which phosphorylates serine 81 on the AR, showed increased expression in sunitinib-resistant models in vivo and in vitro on treatment with sunitinib.

Addition of a proteasome inhibitor to combination enzalutamide and sunitinib treatment abrogated enzalutamide-induced AR degradation in the presence of sunitinib. The addition of the proteasome inhibitor also increased cell proliferation. AR expression was increased by sunitinib treatment in AR-positive cells with transient Speckle-type POZ protein (SPOP) knockdown. However, enzalutamide-induced AR degradation was inhibited in these cells in the presence of sunitinib treatment. Immunoprecipitation showed that ubiquitin associated with AR after treatment with sunitinib in sunitinib-resistant cells. The reduction in AR nuclear translocation caused by enzalutamide was inhibited by competitive binding with dihydrotestosterone.

In vivo, combined sunitinib and enzalutamide treatment delayed time to acquired sunitinib resistance in sunitinib-sensitive, cell-line-derived xenografts. In experimental models with acquired sunitinib resistance, combination treatment inhibited further tumour growth compared with enzalutamide treatment alone. Circulating levels of KLK2 were increased in xenograft models of acquired and intrinsic sunitinib resistance.

In patients, serum KLK2 levels were increased in those whose disease progressed while receiving sunitinib plus dalteparin compared with those who did not experience disease progression.

Overall, these data suggest that the AR is involved in sunitinib resistance in kidney cancer, which can be reversed by combination treatment with an AR antagonist. These data provide a rationale for clinically testing this combination in patients with sunitinib-resistant disease.