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PDK1 Signaling Toward PLK1–MYC Activation Confers Oncogenic Transformation, Tumor-Initiating Cell Activation, and Resistance to Mtor-Targeted Therapy

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PDK1 Signaling Toward PLK1–MYC Activation Confers Oncogenic Transformation, Tumor-Initiating Cell Activation, and Resistance to Mtor-Targeted Therapy Published OnlineFirst July 25, 2013; DOI: 10.1158/2159-8290.CD-12-0595 RESEARCH ARTICLE PDK1 Signaling Toward PLK1–MYC Activation Confers Oncogenic Transformation, Tumor-Initiating Cell Activation, and Resistance to mTOR-Targeted Therapy Jing Tan 1 , Zhimei Li 1 , Puay Leng Lee 1 , Peiyong Guan 2 , Mei Yee Aau 1 , Shuet Theng Lee 1 , Min Feng 1 , Cheryl Zihui Lim 1 , Eric Yong Jing Lee 1 , Zhen Ning Wee 1 , 3 , Yaw Chyn Lim 4 , R.K. Murthy Karuturi 2 , and Qiang Yu 1 , 4 , 5 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst July 25, 2013; DOI: 10.1158/2159-8290.CD-12-0595 ABSTRACT Although 3-phosphoinositide–dependent protein kinase-1 (PDK1) has been pre- dominately linked to the phosphoinositide 3-kinase (PI3K)–AKT pathway, it may also evoke additional signaling outputs to promote tumorigenesis. Here, we report that PDK1 directly induces phosphorylation of Polo-like kinase 1 (PLK1), which in turn induces MYC phosphorylation and protein accumulation. We show that PDK1–PLK1–MYC signaling is critical for cancer cell growth and survival, and small-molecule inhibition of PDK1/PLK1 provides an effective approach for therapeutic targeting of MYC dependency. Intriguingly, PDK1–PLK1–MYC signaling induces an embryonic stem cell–like gene signature associated with aggressive tumor behaviors and is a robust signaling axis driv- ing cancer stem cell (CSC) self-renewal. Finally, we show that a PLK1 inhibitor synergizes with an mTOR inhibitor to induce synergistic antitumor effects in colorectal cancer by antagonizing compensatory MYC induction. These fi ndings identify a novel pathway in human cancer and CSC activation and provide a therapeutic strategy for targeting MYC-associated tumorigenesis and therapeutic resistance. SIGNIFICANCE: This work identifi es PDK1–PLK1–MYC signaling as a new oncogenic pathway driving oncogenic transformation and CSC self-renewal. Targeted inhibition of PDK1/PLK1 is robust in target- ing MYC dependency in cancer cells. Thus, our fi ndings provide important insights into cancer and CSC biology and have signifi cant therapeutic implications. Cancer Discov; 3(10); 1–16. ©2013 AACR. See related commentary by Cunningham and Ruggero, p. xxxx. INTRODUCTION human malignancy can also be caused by gene amplifi cation or abnormal phosphorylation in the cytosol and nucleus, as The phosphoinositide 3-kinase (PI3K)–AKT pathway is in colon cancer and invasive breast cancer ( 4, 5 ). one of the most commonly deregulated signaling pathways One of the most defi ned PDK1 targets relevant in human can- in human cancers ( 1 ). Genetic aberrations affecting this cer is AKT. Specifi cally, PDK1 directly phosphorylates AKT on pathway, such as activating mutations of PIK3CA or inacti- T308, but requires mTOC complex 2 (mTORC2)-induced AKT vation of PTEN, have been identifi ed in virtually all epithe- phosphorylation on S473 to confer a full activation ( 6 ). Given lial tumors ( 2 ). The 3-phosphoinositide–dependent protein its connection to AKT, PDK1 has been pursued as a critical kinase-1 (PDK1) is known to be activated as a result of anticancer target ( 7 ). However , in view of the diversity of PDK1 the accumulation of the PI3K product phosphatidylinositol- substrates, additional downstream targets of PDK1 may con- 3,4,5-trisphosphate (PIP3), and thus considered an important fer aberrant signaling heterogeneity and complexity in human component of the PI3K pathway. PDK1 is a master regula- malignancy. Indeed, it has been recently shown that inhibition tor of AGC kinase members, including AKT, p70 ribosomal of PDK1 has no signifi cant effect on AKT signaling in a PTEN- S6 kinase (S6K), serum- and glucocorticoid-induced protein defi cient transgenic tumor mouse model ( 8 ) or breast tumor kinase (SGK), and protein kinase C (PKC) family members, growth ( 9 ), and oncogenic functions of PDK1 through sub- thus having multiple roles in various physiologic processes strates other than AKT, such as SGK3 ( 10 ), mitogen- activated such as metabolism, growth, proliferation, and survival ( 3 ). protein kinase (MAPK; ref. 11 ), or PKCα ( 12 ), have also been In human cancers, PDK1 is thought to be constitutively acti- reported. In addition, our recent work has shown that PDK1 vated upon elevation of PIP3 owing to the loss of PTEN or is required for MYC protein accumulation in colon cancer cells gain of PIK3CA activity. In addition, PDK1 deregulation in treated with the mTOR inhibitor rapamycin ( 5 ), indicating a potential functional link of PDK1 with MYC in oncogenesis. MYC is implicated in both cancer and stem cell self-renewal. Authors’ Affi liations: 1 Cancer Therapeutics and Stratifi ed Oncology, 2 Information and Mathematical Science, Genome Institute of Singapore, The relationship between stem cells and human cancers has Agency for Science, Technology and Research (A*STAR), Biopolis; 3 Gradu- become an important issue in cancer research given that self- ate School for Integrative Sciences and Engineering; 4 Department of Phys- renewal is a hallmark of both cell types ( 13 ). Genes associated iology, Yong Loo Lin School of Medicine, National University of Singapore; with embryonic stem cell (ESC) identity, including pluripotency 5 and Cancer and Stem Cell Biology, DUKE-NUS Graduate Medical School transcription factors, Polycomb targets, and MYC targets, have of Singapore, Singapore. been observed in aggressive human cancers and are associated Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). with poor disease outcome ( 14 ). Moreover, the MYC- associated molecular network is strikingly similar between ESC and Corresponding Author: Qiang Yu, Cancer Therapeutics and Stratifi ed Oncol- ogy, Genome Institute of Singapore, Agency for Science, Technology and human cancer transcription programs ( 15 ), and ectopic overex- Research (A*STAR), 60 Biopolis Street, Singapore. Phone: 65-6808-8127; pression of MYC in differentiated somatic cells can induce both Fax: 65-6808-9003; E-mail: [email protected] an ESC gene signature and properties of cancer stem cells (CSC; doi: 10.1158/2159-8290.CD-12-0595 ref. 16 ). These fi ndings suggest that activation of an ESC-like © 2013 American Association for Cancer Research. gene expression program in adult cells may confer self-renewal OCTOBER 2013CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst July 25, 2013; DOI: 10.1158/2159-8290.CD-12-0595 RESEARCH ARTICLE Tan et al. to cancer cells or CSCs. Notably, although the cancer-associated of MYC resulted in much-reduced transformation of HEK- ESC-like gene regulation by transcription factors has been well PDK1 cells, but not of HEK-E545K cells ( Fig. 1C ), showing a documented, its regulation by a druggable kinase-driven signal- MYC dependency for PDK1-induced transformation. Moreo- ing pathway has yet to be identifi ed. ver, in a series of dose–response analysis (see Supplementary In the present study, we investigated PDK1-evoked key Fig. S1D and S1E), HEK-PDK1 cells, compared with HEK- signaling events required for oncogenic transformation. We E545K cells, were much more sensitive to small-molecule PDK1 identifi ed that the PDK1–Polo-like kinase 1 (PLK1)–MYC inhibitors BX795 and BX912 ( Fig. 1D , left and Supplementary pathway is a major driver pathway conferring PDK-induced Fig. S1D). In contrast, E545K-transformed cells were much transformation, and its existence is readily evident in human more sensitive to the PI3K inhibitor GDC-0941 and the AKT cancers. We further show that PDK1–PLK1–MYC signal- inhibitors MK2206 and GSK690693 ( Fig. 1D , left and Sup- ing drives an ESC-like gene expression signature relevant in plementary Fig. S1E). Consistent with these effects, BX795 human cancers and is robust in inducing a CSC phenotype. It reduced MYC accumulation but had only a modest effect is also involved in resistance to mTOR inhibitor in colorectal on AKT. In contrast, GDC-0941 or MK2206 easily abolished cancer cells. These fi ndings provide important insights into phosphorylations of AKT in HEK-E545K cells, but had no such cancer, CSC biology, and potential new treatment for target- effects on MYC inhibition ( Fig. 1D , right). These results showed ing MYC dependency in human cancers. the differential pathway dependency for the two transformed cell systems. Interestingly, MYC-transformed cells were also RESULTS sensitive to BX795 ( Fig. 1D , left), which is consistent with the observation that BX795 was able to eliminate the exogenous PDK1-Induced MYC Protein Induction Confers MYC in these cells ( Fig. 1D , right). Altogether, these data show Oncogenic Transformation that PDK1-induced transformation depends more on MYC, As the fi rst step in investigating the differential signal- but less on AKT signaling, when compared with E545K-driven ing pathways activated by PDK1 or PI3K in tumorigenesis, transformation. The data also suggest that MYC-dependent we compared the transforming capacity of PDK1 and PI3K cells become sensitive to the PDK1 inhibitor, regardless of by using the in vitro transformation assay that measures PDK1 status, which reveals a PDK1 dependency in MYC-driven anchorage-independent growth in soft agar. We began with cells. PDK1-induced MYC activation upon transformation was semitransformed human embryonic kidney epithelial cells also observed in immortalized human mammary epithelial cells (HEK) that express a low level of activated HRASV12 (HEK- (HMEC) and prostate epithelial cells (RWPE-1; Fig. 1E and F ), TERV; ref. 17 ) and infected them with retroviral vectors suggesting that MYC activation by PDK1 is not restricted to expressing PDK1, MYC, a constitutively activating mutant HEK cells but occurs in multiple epithelial lineages. of PIK3CA (E545K), or PTEN short hairpin RNA (shRNA), To show the physiologic relevance of the PDK1–MYC con- resulting in stable cell lines designated as HEK-PDK1, HEK- nection in human cancers, we showed that PDK1 knockdown MYC, HEK-E545K, or HEK-sh PTEN cells, respectively.
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