Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase C␥-protein kinase C (PLC␥-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/ Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.
By behaving as molecular hubs, scaffold proteins can assemble a large number of signaling molecules and organize complicated intracellular signaling networks in time and space. Owing to their crucial role in mediating intracellular signaling related to tumor cell growth and migration, recent studies have highlighted the relevance of scaffold proteins in human cancers and indicated that interfering with their expression and/or their ability to bind effector proteins can inhibit cancer progression. Here, we show that receptor for activated C-kinase 1 (RACK1), a ubiquitously expressed scaffolding protein, plays a crucial regulatory role in tumor growth. Using an RNA silencing approach, we found that downregulation of RACK1 expression in HeLa and A673 tumor cells markedly suppressed the proliferation and invasion of these cells in vitro and tumor development in vivo. Consequently, we found that significant suppression of constitutive phosphorylation of Akt and MAPK by RACK1 silencing may contribute to the inhibition of tumor growth. Moreover, RACK1 silencing significantly attenuated tumor-associated angiogenesis by, at least in part, inhibiting the expression of two critical angiogenic factors, namely vascular endothelial growth factor-B and fibroblast growth factor 2. The results of the present study show that RACK1 is a potent enhancer of tumor growth and, thus, a potential anti-cancer therapeutic target. (Cancer Sci
Pharbin, a 5-phosphatase that induces arborization, is one of the phosphoinositide 5-phosphatases that is highly mutated in patients with Joubert syndrome. Pharbin can hydrolyse PI(4,5)P(2) and PI(3,4,5)P(3) and has the same substrate specificity as SHIP2 and SKIP, which negatively regulate PI3K signalling. Here, we investigated the role of pharbin in IGF-1/PI3K signalling. Ectopic expression of pharbin markedly suppressed the IGF-1-induced activation of Akt without affecting p42/44 MAP kinase phosphorylation. In contrast, pharbin silencing by RNA interference increased the IGF-1-induced phosphorylation of Akt, suggesting that pharbin negatively regulates PI3K/Akt signalling. Pharbin expression also inhibited the phosphorylation of p70 S6 kinase and 4E-BP1 as well as the subsequent protein synthesis in response to IGF-1 treatment. Taken together, these results indicate that pharbin is an important negative regulator of IGF-1/PI3K/Akt signalling and protein synthesis.
Anti-angiogenesis is an effective cancer treatment, however, tumor progression and refractoriness to anti-angiogenesis are observed in both clinical and preclinical cancer models. Stress under hypoxia and nutrition deprivation might play an important role in the tumor aggressiveness and refractoriness. Here we show that a long term hypoxia and nutrient starvation double-deprivation stress (DDS) stimulates migration, invasion and anchorage independent growth of tumor cells in vitro and an increased tumor progression and angiogenesis in vivo associated with an increase in blood vessels and infiltration of CD11b+ cells. We also observed up-regulation of DDS-responsive genes including VEGF-A, FGF18, and JHDM2A. We found that histone demethylase JHDM2A is commonly up-regulated under DDS in human and mouse cancer cell lines in vitro and in vivo at the phase of pre-angiogenic switch and refractory phase of anti-angiogenic treatments. Specific inhibition of JHDM2A by siRNAs significantly suppressed tumor growth in vivo. Importantly, suppression of JHDM2A demonstrated synergetic effects in treatment with anti-VEGF neutralizing antibody (bevacizumab) or small molecular inhibitor for VEGF receptors (sunitinib), and improved an efficacy of anti-angiogenesis. Collectively, our results indicate that minor population of cancer cells resistant to both hypoxia and low nutrition acquires tumor aggressiveness through specific mechanisms upon the DDS responses. Our observations suggest that the targeting of cancer cells resistant to hypoxia and nutrient starvation by inhibition of DDS-responsible genes such as JHDM2A can be utilized for a novel treatment to overcome the refractoriness to anti-angiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4270. doi:10.1158/1538-7445.AM2011-4270
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