SPOP Promotes GLI3‐dependent SHH Signaling in Breast Cancer

Abstract

Personalized treatment, which is designed to only block oncogenic pathways activated by specific cancer mutations, is rapidly rising as a superior treatment for cancer. One gene that has recently been identified as highly mutated in a variety of cancers is Speckle Type POZ protein (SPOP), a substrate binding subunit of an E3 ubiquitin ligase that has been shown to target Sonic Hedgehog (SHH) downstream effectors for degradation. Interestingly, recent studies have found that SPOP is downregulated in up to 70% of breast cancer tumors and, furthermore, a correlation has been found between high levels of SHH signaling and poor prognostic pathological breast cancer features. Previous studies from our laboratory have shown that SPOP binds directly to the SHH downstream effector GLI3 to target it for degradation in a manner that is disrupted by prostate cancer-associated SPOP mutations. Since SHH signaling has been shown to be a key player in breast cancer oncogenesis, it is likely that SPOP plays a similar role in breast cancer progression. We therefore hypothesize that downregulation of SPOP induces hyper-activated SHH signaling to promote breast cancer progression through stabilization of GLI3. In order to study this, we employed a lentivirus-mediated approach to generate SPOP knockdown breast cancer cells. Next, we carried out western blot analysis to examine GLI3 protein levels when SPOP is downregulated followed by co-immunoprecipitation analysis to examine a GLI3/SPOP interaction. Finally, we used MG132 treatment to block the proteasome pathway in order to determine whether SPOP-mediated degradation of GLI3 functions through the proteasome. Our results indicate that SPOP knockdown promotes upregulation of the SHH downstream effector GLI3 through a direct physical interaction. Furthermore, we show that SPOP likely targets GLI3 for degradation through the proteasome pathway as MG132 treatment promotes GLI3 stabilization even in the presence of SPOP. Our collective results therefore indicate that SPOP targets GLI3 for degradation through the proteasome pathway which, in turn, would cause hyper-activated SHH signaling and subsequent progression of breast cancer oncogenesis.