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t Agar and Tumor Growth Because it has been shown that PDK1 protein and mRNA are overexpressed in a majority of human breast cancers, we assessed the tumorigenic effect Beta-Lapachone of PDK1 overexpression in both MDA MB 231 and T 47D . The addition of exogenous PDK1 substantially elevated the number of colonies grown within the soft agar . We next determined no matter whether this in vitro–enhanced tumorigenicity resulted in Beta-Lapachone a tumor growth increase. PDK1 overexpressing MDA MB 231 cells, subcutaneously injected in mice, formed tumors having a substantially larger volume than those of cells transduced with all the empty vector . Accordingly, tumors originating from PDK1 overexpressing cells displayed a decreased number of apoptotic cells and an increase in proliferating cells, statistically significant only within the central region on the tumors .
The Kinase Activity of PDK1 Is Needed to Regulate Tumor Growth To understand the molecular mechanism activated by PDK1 in the course of anchorage independent and tumor growth, we investigated which activity of PDK1 is essential for this function. To achieve this objective, cells, downregulated for PDK1, were transduced with lentiviral vectors expressing PDK1 mutants which are insensitive Lomeguatrib to gene silencing. The following cDNAs were expressed in MDA MB 231: PDK1 wild type , K110N mutant that abolishes kinase activity , and PH domain–deleted mutant that impedes binding to PIP3 at the membrane . The introduction of PDK1 into silenced cells was able to recover the capability to grow in soft agar, whereas the PDK1 KD was unable to rescue the phenotype, suggesting that kinase activity is essential for tumorigenesis.
On the contrary, PDK1 mutant within the PH domain was able to rescue the anchorage independent growth . To further assistance the involvement of PDK1 kinase activity in soft agar growth and anoikis, we utilised two kinase inhibitors of PDK1: BX 795 and OSU 03012. BX 795 inhibited soft agar growth very effectively and promoted anoikis . Notably, Carcinoid BX 795 was far more efficient in inducing apoptosis when cells were grown within the absence of adhesion than once they were plated on plastic . Equivalent results were obtained with OSU 03012 . Although these chemical compounds usually are not distinct inhibitors for PDK1, their EC50 concentration was sensitive to PDK1 expression levels. The truth is, PDK1 silencing sensitized apoptosis induced by BX 795, by lowering the EC50 to 3.
80 × 10−6 M, whereas PDK1 overexpression produced them far more resistant with EC50 _ 4. 30 × 10−5M. To assess no matter whether the PKD1 kinase activity was also essential for tumor growth, we subcutaneously injected silenced cells Lomeguatrib transduced with PDK1 or PDK1 KD. The reintroduction of PDK1 induced the formation of tumors similar to controls, whereas the expression of PDK1 KD mutant was completely unable to rescue the phenotype . Furthermore, PDK1 reexpression restored the percentage of Ki 67–positive cells within the central region on the tumor , whereas it decreased the number of apoptotic cells . Akt Phosphorylation Is not Affected by PDK1 Down regulation To further evaluate PDK1 kinase activity arising fromreintroduction of PDK1 mutants, we analyzed Akt1 phosphorylation on Thr308 following stimulation with hEGF.
Unexpectedly, the low levels of PDK1 remaining following gene silencing were nonetheless adequate to phosphorylate Akt at the exact same extent of control cells . Nevertheless, PDK1 reexpression, which truly elevated PDK1 expression above its physiological levels, led to an increase in Beta-Lapachone Akt Thr308 phosphorylation, which was prevented by inactivating mutations within the PDK1 kinase domain . Equivalent effects were observed on phospho Ser473 Akt. The Akt phosphorylation trend was paralleled by the phosphorylation of Akt downstream effectors. PDK1 knockdown was unable to impair the phosphorylation of both GSK3B and FOXO, and PDK1 overexpression caused an elevated phosphorylation, which was not observed in cells expressing PDK1 kinase dead .
The addition of PI3K inhibitor, prior to the hEGF stimulation, totally abolished both FOXO and Akt phosphorylation, whereas it was ineffective in inhibiting Lomeguatrib PDK1 and GSK3B phosphorylation. Then, we extended the Akt phosphorylation analysis in tumors of MDA MB 231 cells. The confocal microscopy analysis revealed that phosphorylation of Beta-Lapachone Thr308 of Akt was unchanged on PDK1 silencing. In this case, PDK1 reexpression was unable to increase Akt phosphorylation in tumors . Nevertheless, levels of PDK1 and phospho Ser241 PDK1 were modest in shPDK1#79 compared with those Lomeguatrib in shScr tumors, whereas levels were far more evident in tumors in which PDK1 was reexpressed. In contrast, PDK1 KD tumors exhibited low levels of PDK1 phosphorylation on Ser241, as expected within the case of autophosphorylation . PDK1 Tumorigenesis Is Akt Independent Given that PDK1 kinase activity was necessary for both cell anchorage– independent and tumor growth, despite the fact that its major substrate, Akt, was not differentially phosphorylated in PDK1 knockdown cells, we decided to unravel the functional role of Akt in PDK1 mediated tumor