Previous efforts to develop drugs that directly inhibit the activity of mutant protooncogene that occur in 30% of human cancers and are particularly prevalent in adenocarcinomas of the pancreas, lung, and colon (Karnoub and Weinberg, 2008). downstream of KRAS, and several signaling molecules that are regulated by RAS, such as RAF, MEK, and PI3K, are being targeted by drugs in early clinical trials (Lim and Counter, 2005; Gupta et al., 2007; Engelman et al., 2008; Yu et al., 2008; Wee et al., 2009; Halilovic et al., 2010). In addition to these efforts, which build on previous insights into the linear signaling pathways through which RAS promotes cellular viability and proliferation, several studies have used large-scale functional genomic screens to search for genes that are aberrantly required as a result of adaptation to a transforming KRAS mutation and might therefore represent new therapeutic targets Phenylpiracetam IC50 (Barbie et al., 2009; Luo et al., 2009; Scholl et al., 2009; Wang et al., 2010; Vicent et al., 2010). Using high-throughput RNA interference (RNAi), we recently described that the expression Phenylpiracetam IC50 of a functionally uncharacterized serine/threonine kinase, STK33, is required by human cancer cells that are dependent on mutant KRAS, but not untransformed cells or cancer cells with a different oncogenic lesion (Scholl et al., 2009). Although the role of STK33 in normal cellular physiology and in KRAS mutant cancer cells is not well understood, the enhanced STK33 dependence of KRAS mutant cells supports STK33 as an attractive target for therapy that could be pursued with drug discovery approaches. However, to inform this strategy, additional studies are necessary to better understand the functional link between mutant KRAS and STK33 and to elucidate the mechanism through which STK33 promotes cancer cell viability. The primary goal of this study was to gain insight into the signaling pathways through which STK33 functions in human cancer cells. Using mass spectrometryCbased proteomics, we observed that STK33 physically interacts with components of the HSP90 chaperone complex that is essential for the proper folding, stabilization, and activation of numerous proteins involved in cell survival and proliferation (Picard, 2002; Taipale et al., 2010), including oncoproteins that are mutated or Phenylpiracetam IC50 overexpressed in certain cancer types (Gorre et al., 2002; George et al., 2004; Sawai et al., 2008; Cerchietti et al., 2009; Marubayashi et al., 2010). Genetic or pharmacologic inhibition of HSP90 in human cancer cell lines of various tissue origin induced proteasome-mediated degradation of STK33, resulting in apoptosis, both in vitro and in xenotransplant tumors, preferentially in cells harboring mutant KRAS. Furthermore, cells derived from KRAS mutant primary human colon carcinomas were significantly more sensitive to HSP90 inhibitor treatment. These findings identify STK33 as a new HSP90 client protein and provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells that has been noted before but remained unexplained until now (Wong et Rabbit polyclonal to OX40 al., 2011; West et al., 2011; Sos et al., 2009). Furthermore, the data indicate that the requirement for STK33 may be exploited to target mutant KRAS-driven cancers, and suggest a restorative strategy that could become evaluated immediately because HSP90 inhibitors are currently undergoing medical evaluation in individuals with numerous malignancies. Finally, these results display that the ideal use of HSP90 inhibitors will depend on understanding the practical dependencies of specific cancers, and support KRAS mutation status as a marker for predicting responsiveness to these providers. RESULTS HSP90 binds to and stabilizes STK33 in human being malignancy cells We used a mass spectrometryCbased approach to determine STK33 protein connection partners in human being malignancy cells. The breast malignancy cell lines MDA-MB-231 (harboring Phenylpiracetam IC50 a KRASG13D mutation) and BT-20 (harboring WT KRAS) were stably transduced with a lentiviral vector encoding Flag-tagged STK33 or an bare control vector. Protein lysates Phenylpiracetam IC50 of these cell lines were incubated with anti-Flag agarose, and separated healthy proteins were separated by PAGE (Fig. 1 a). Each lane was excised and divided into 10 equally sized items, and peptides were sequenced by microcapillary reverse-phase HPLC nanoelectrospray tandem mass spectrometry. The most highly enriched proteins in the STK33-conveying samples were two users of the HSP90 family of chaperones, HSP90AM1 (also known as HSP90B) and HSP90AA1 (also known as HSP90A). In addition, the HSP90 adaptor protein CDC37 was also significantly overrepresented in the STK33-conveying samples (Fig. 1 b). Coimmunoprecipitation (coIP) tests with MDA-MB-231 cells stably conveying hemagglutinin (HA)-labeled STK33 confirmed the joining of STK33 to HSP90 and CDC37 (Fig. 1 c). Number 1. HSP90 acquaintances with and stabilizes STK33. (a) Anti-Flag IPs were performed with KRAS WT BT-20 and KRAS mutant MDA-MB-231 breast malignancy cell lines stably transduced with bare vector (EV), N-terminally Flag-tagged STK33 (Flag-STK33), or C-terminally … HSP90 is definitely known to strengthen and activate multiple proteins (so-called clients; Picard, 2002; Taipale et al., 2010), several of.