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. 2012 Aug;14(8):719-31.
doi: 10.1593/neo.12856.

3-phosphoinositide-dependent kinase 1 controls breast tumor growth in a kinase-dependent but Akt-independent manner

Paolo Armando Gagliardi  1 , Laura di Blasio, Francesca Orso, Giorgio Seano, Roberto Sessa, Daniela Taverna, Federico Bussolino, Luca Primo
Affiliations

3-phosphoinositide-dependent kinase 1 controls breast tumor growth in a kinase-dependent but Akt-independent manner

Paolo Armando Gagliardi et al. Neoplasia. 2012 Aug.

Abstract

3-phosphoinositide-dependent protein kinase 1 (PDK1) is the pivotal element of the phosphatidylinositol 3 kinase (PI3K) signaling pathway because it phosphorylates Akt/PKB through interactions with phosphatidylinositol 3,4,5 phosphate. Recent data indicate that PDK1 is overexpressed in many breast carcinomas and that alterations of PDK1 are critical in the context of oncogenic PI3K activation. However, the role of PDK1 in tumor progression is still controversial. Here, we show that PDK1 is required for anchorage-independent and xenograft growth of breast cancer cells harboring either PI3KCA or KRAS mutations. In fact, PDK1 silencing leads to increased anoikis, reduced soft agar growth, and pronounced apoptosis inside tumors. Interestingly, these phenotypes are reverted by PDK1 wild-type but not kinase-dead mutant, suggesting a relevant role of PDK1 kinase activity, even if PDK1 is not relevant for Akt activation here. Indeed, the expression of constitutively active forms of Akt in PDK1 knockdown cells is unable to rescue the anchorage-independent growth. In addition, Akt down-regulation and pharmacological inhibition do not inhibit the effects of PDK1 overexpression. In summary, these results suggest that PDK1 may contribute to breast cancer, even in the absence of PI3K oncogenic mutations and through both Akt-dependent and Akt-independent mechanisms.

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Figures

Figure 1
Figure 1
PDK1 down-regulation reduces anchorage-independent growth and increases apoptosis. MDA-MB-231 and T-47D cells were transduced with a scramble shRNA (shScr), two separate PDK1-_targeting shRNAs (shPDK1#79 and shPDK1#81), or untransduced (uninfected) and were used for the indicated investigations: (A and B) Protein lysates were obtained and used in immunoblot analysis for PDK1. Tubulin was used as a loading control. (C and D) Soft agar colony formation assay. Only colonies larger than 100 µm diameter were counted. (E and F) Apoptosis assay of cells cultured for 24 hours on plastic (gray bar) or maintained in suspension (black). The number of apoptotic cells was evaluated by using the anti-cytokeratin 18 fragment antibody. *P < .05, **P < .01, ***P < .001.
Figure 2
Figure 2
PDK1 knockdown impairs tumor formation. (A) MDA-MB-231 or (B) LM2-4175 cells transduced with PDK1-_targeting shRNAs (shPDK1#79 and shPDK1#81) or with a scramble sequence (shScr) were injected subcutaneously in immunodeficient mice. Tumor weight was evaluated 4 weeks after injection and expressed in milligrams. Red line indicates the average tumor weight. N = number of mice. (C) Apoptosis within tumors originating from MDA-MB-231 cells, transduced as in A, was evaluated using TUNEL assay, and representative images are shown in false colors: black/blue, low fluorescence intensity; white, high intensity. Quantification of apoptosis (D) and proliferating Ki-67-positive cells (E) in three different regions of tumors as in A. The percentage of apoptotic and proliferating cells was evaluated in the peripheral, intermediate, and central regions of the tumors. *P < .05, **P < .01, ***P < .001.
Figure 3
Figure 3
PDK1 overexpression increases anchorage-independent growth and tumor formation. (A and B) Expression of PDK1 was evaluated by immunoblot in MDA-MB-231 or T-47D cells transduced with PDK1 or with an empty vector. Tubulin was used as loading control. (C and D) Soft agar colony formation assay of MDA-MB-231 and T-47D cells transduced as indicated in A and B. Only colonies larger than 100 µm diameter were counted. (E) MDA-MB-231 cells transduced as in A were injected subcutaneously in immunodeficient mice. Tumor weight was evaluated 4 weeks after injection and expressed in milligrams (mg). Red line indicates the average tumor weight. N = number of mice. (F) Quantification of apoptosis in three different regions of the tumors referring to E. The percentage of apoptotic cells was evaluated in the peripheral, intermediate, and central regions of the tumors. *P <.05, **P <.01, ***P <.001.
Figure 4
Figure 4
PDK1 regulates anchorage-independent and tumor growth by a kinase-dependent mechanism. (A) MDA-MB-231 PDK1 knockdown cells (shPDK1#79) as in Figure 1 were transduced with vectors carrying PDK1 wild-type (PDK1), PDK1 kinase-dead (PDK1 KD), PDK1 PH-domain-deleted (PDK1 ΔPH), or empty vectors. PDK1 expression levels are shown in Figure W4. Soft agar colony formation assay was performed with the various cell lines and only colonies larger than 100 µm diameter were counted. (B) Soft agar colony formation assay with MDA-MB-231 cells was measured in the presence of different concentrations of the PDK1 inhibitor BX-795. EC50 is indicated. (C) Apoptosis assay with MDA-MB-231 cultured on plastic (gray bar) or maintained in suspension (black) for 24 hours and treated with different concentrations (M) of the PDK1 inhibitor BX-795. The number of apoptotic cells was measured considering the cytokeratin 18 fragment-positive cells and the reported EC50. (D) MDA-MB-231 cells transduced with scramble shRNA (shScr) or with PDK1-_targeting shRNAs (shPDK1#79) and retransduced with PDK1 wild-type (PDK1), PDK1 kinase-dead (PDK1-KD), or empty vector were injected subcutaneously in immunodeficient mice. Tumor weight was evaluated 4 weeks after injection and expressed in milligrams (mg). Red line indicates the average tumor weight. *P <.05, **P < .01, ***P < .001.
Figure 5
Figure 5
PDK1 knockdown does not affect Akt phosphorylation. (A) Immunoblot analysis with the indicated antibodies was performed on lysates of MDA-MB-231 cells transduced with scramble shRNA (shScr) or PDK1-_targeting shRNAs (shPDK1#79) and retransduced with PDK1 wild-type (PDK1), PDK1 kinase-dead (PDK1-KD), or empty vectors. Cells were treated or not with PI3K inhibitor then stimulated with different concentrations of hEGF. (B) Immunostaining and confocal microscopy analysis of tumor sections originating from MDA-MB-231 transduced as indicated in A. The images are representatives of four tumors and were acquired with the same confocal parameters and magnification. Top panels are stained with anti-phosphoPDK1 (pS241PDK1; red) and anti-PDK1 (PDK1; green). Bottom panels are stained with anti-phosphoAkt (pT308Akt; red) and anti-Akt1 (Akt1; green).
Figure 6
Figure 6
Akt overexpression does not rescue anchorage-independent growth of PDK1 knockdown cells. (A) Immunoblot analysis for the indicated antibodies was performed on lysates of MDA-MB-231 cells transduced with scramble shRNA (shScr) or with PDK1-_targeting shRNAs (shPDK1#79) and retransduced with Akt1 or empty vectors. β-Actin was used as loading control. (B) Soft agar colony formation assay performed on MDA-MB-231 cells transduced as indicated. Only colonies larger than 100 µm diameter were counted. (C) Immunoblot analysis of lysates of MDA-MB-231 cells transduced with scramble shRNA (shScr) or with PDK1-_targeting shRNAs (shPDK1#79) and retransduced with PDK1 wild-type (PDK1), different active mutants of Akt (myr-Akt1, myr-Akt2, Akt1DD), inactive mutant of Akt (myr-Akt1 K179M), or empty vectors. (D) Soft agar colony formation assay performed on MDA-MB-231 cells transduced as indicated. Colonies larger than 100 µm diameter were counted. ***P <.001.
Figure 7
Figure 7
Akt down-regulation and inhibition does not reduce anchorage-independent growth of PDK1-overexpressing MDA-MB-231. (A and B) PDK1-overexpressing MDA-MB-231 and T-47D cells were transduced with a scramble shRNA (shScr), two different Akt1 (shAkt1#86 and shAkt1#97) or Akt2 (shAkt2#17 and shAkt2#68) _targeting shRNAs, lysed, and analyzed by immunoblot with the indicated antibodies. (C and D) Soft agar colony formation assay performed on MDA-MB-231 and T-47D cells transduced as indicated above. Only colonies larger than 100 µm diameter were counted. (E and F) Soft agar colony formation assay of MDA-MB-231 and T-47D cells transduced with PDK1 or empty vectors and treated with different concentrations of the Akt inhibitor VIII, PDK1 inhibitor, BX-795. EC50 is indicated. (G and H) Soft agar colony formation assay of MDA-MB-231 and T-47D cells transduced with PDK1 or empty vectors and treated with different concentrations of PDK1 inhibitor, BX-795. EC50 is indicated. *P <.05, **P <.01, ***P <.001.
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