Parathyroid hormone-related protein induces cell survival in human renal cell carcinoma through the PI3K Akt pathway: evidence for a critical role for integrin-linked kinase and nuclear factor kappa B

Abstract

We have recently shown that parathyroid hormone-related protein (PTHrP), a cytokine-like polyprotein, is critical for human renal cell carcinoma (RCC) growth by inhibiting tumor cell apoptosis. Here, we have explored mechanisms by which PTHrP controls tumor cell survival. Using specific inhibitors of phosphoinositide 3-kinase (PI3K) and depletion of Akt kinase by RNA interference, we established that PTHrP is one of the main factor involved in the constitutive activation of this pathway in human RCC, independently of von Hippel-Lindau (VHL) tumor suppressor gene expression. Interestingly, PTHrP induced phosphorylation of Akt at S473 but had no influence on phosphorylation at T308. Through transfection with integrin-linked kinase (ILK) constructs and RNA interference, we provide evidence that ILK is involved in human RCC cell survival. PTHrP activates ILK which then acts as a phosphoinositide-dependent kinase (PDK)-2 or a facilitator protein to phosphorylate Akt at S473. Among other kinases tested, only ILK was shown to exert this function in RCC. Using specific inhibitors, western blot and transcription assay, we identified nuclear factor kappa B (NF-kappaB) as the downstream Akt target regulated by PTHrP. Since RCC remains refractory to current therapies, our results establish that the PI3K/ILK/Akt/NF-kappaB axis is a promising target for therapeutic intervention.

LIM-class homeobox gene Lim1, a novel oncogene in human renal cell carcinoma

“…Cell density was assessed either alone or in combination with cyclopamine (LC Laboratories, Woburn, MA, USA), SB216763 (glycogen synthase kinase-3 inhibitor, SigmaAldrich), LY294002 (PI3K inhibitor, Sigma-Aldrich), BAY 11-7085 (NF-B inhibitor, Calbiochem, Fontenay-sous-Bois, France), or U0126 (MAPK inhibitor, Calbiochem). We have previously shown the specificity of LY294002, BAY11-7085 and cyclopamine on their specific targets at the concentration used by western blot (Sourbier et al, 2006Agouni et al, 2007;Dormoy et al, 2009). The specificity at the concentration of the other inhibitors has been shown in numerous studies.…”

Parathyroid hormone (PTH) rapidly enhances CFTR-mediated HCO3− secretion in intestinal epithelium-like Caco-2 monolayer: a novel ion regulatory action of PTH

“…Impedance spectroscopy was performed by using 1255B frequency response analyzer and 1287A electrochemical interface (Solartron, Farnborough, UK). Sine-wave alternating current (35 A/cm 2 root mean square amplitude, 0.5 Hz-10 kHz) was applied across Caco-2 monolayer at different time points, i.e., before adding 10 nmol/l PTH 1-34 or 10 mol/l forskolin, and at 1,5,10,15,20,25,30,35 (Fig. 1A) was automatically analyzed by Zplot/Zview.…”

“…Although PTH is known to exert its actions mainly through the cAMP-dependent PKA pathway, other signaling pathways, namely, Ca 2ϩ -dependent PKC and PI3K, have been reported to mediate PTH actions in the renal proximal tubular epithelium (1,16,38) and intestinal epithelium (23), respectively. In the present study, Caco-2 monolayers were exposed on both sides to inhibitors of cAMP/ PKA, Ca 2ϩ /PKC or PI3K pathways prior to PTH 1-34 stimulation.…”

Hormone signaling pathways as treatment targets in renal cell cancer (Review)

“…The indirect antitumor effects of SSTRs include anti-angiogenic actions (22,23). Moreover, parathyroid hormone-related protein (PTHrP), a cytokine-like polyprotein, was determined to be a survival factor for human ccRCC: its expression was found to be negatively regulated by the VHL tumor suppressor gene at the level of messenger RNA (mRNA) stability (9), and induced phosphorylation of Akt at S473 via activated integrin-linked kinase (ILK), which in turn acted as either a phosphoinositide-dependent kinase (PDK2) or a facilitator protein to phosphorylate Akt, with nuclear factor kappa B (NF-κB) serving as the downstream Akt target (24). The examples mentioned above underscore the possibility of alternative pathway activation in ccRCC via hormones and illustrate the need to explore the molecular basis of phenomena observed in clinics (25,26).…”