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

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 driving 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 targeting 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; 3Gradu- 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

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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. The was able to eliminate MYC expression in a variety of human transformation assay results showed that they were all able cancer cell lines ( Fig. 1G ). Moreover, in a panel of breast can- to induce cellular transformation, although PDK1- and cer cell lines in which the MYC-dependent viability has been MYC-induced colonies appeared to be larger in size as previously characterized (18 ), BX795 treatment resulted in compared with that of E545K- or shPTEN-expressing cells similar MYC depletion in all these cells (Fig. 1H ) but prefer- (Fig. 1A and Supplementary Fig. S1A). Consistent with our entially reduced the cell viability of MYC-dependent breast previous report showing a posttranslational MYC induc- cancer cell lines (MDA-MB-231, Hs578T, and SUM159PT) tion by PDK1 (5 ), we detected a marked protein accumula- as compared with the MYC-independent breast cancer cell tion of MYC in HEK-PDK1 cells but not in HEK-E545K lines (T47D and BT474; Fig. 1I ). Of note, in these cell lines or HEK-shPTEN cells (Fig. 1B ), which was not due to the BX795 seemed to inhibit AKT and FOXO3A phosphoryla- induction of Myc mRNA levels (Supplementary Fig. S1B). tions in a cell-dependent manner ( Fig. 1H ). Taken together, We also showed that the kinase activity of PDK1 is required these results show a potential role of PDK1 activity toward for transformation as well as MYC protein induction, as a MYC regulation, which is therapeutically implicated for kinase-dead mutant of PDK1 (PDK1 K100N; ref. 9 ) induced MYC-driven tumors. neither the transformation nor the MYC accumulation (Supplementary Fig. S1C). Synthetic Lethal Screening Identifi es PLK1 as a A survey of known AGC substrates of PDK1 revealed that Crucial Downstream Effector of PDK1 for MYC PDK1 also induced a strong phosphorylation of PKCδ and Induction and Cancer Cell Survival a modest increase of phosphorylated AKT (T308). The other To investigate whether or not there are downstream kinases known PDK1 substrates, including SGK1/3 and S6K, were not of PDK1 that are crucial for MYC induction and cell transfor- activated, nor AKT phosphorylated at S473, which is required mation, we conducted a screen for kinases that, when phar- for a full activation of AKT. In contrast, E545K overexpres- macologically inhibited, selectively kill PDK1-transformed sion induced strong phosphorylation of AKT (at both T308 cells. Among 60 small-molecule protein kinase inhibitors we and S473) as well as the downstream substrates FOXO1 and have screened, we found that two PLK1 inhibitors (BI2536 FOXO3A (Fig. 1B ). Thus, the remarkable observation that and GW843682X), one mitogen-activated protein (MAP)- PDK1 induces transformation in the presence of a weak AKT extracellular signal-regulated kinase (ERK) (MEK) inhibitor activation suggests a potential more functional role of MYC in (PD0325901), one ALK inhibitor (NVP-TAE684), one BRC– this process. Indeed, RNA interference–mediated knockdown ABL inhibitor (PD180970), and one tyrosine kinase inhibitor

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PDK1–PLK1–MYC Signaling in Transformation, Cancer Stem Cells, and Drug Resistance RESEARCH ARTICLE

A CD siNC 120 PDK1 120 siMyc E545K PDK1 MYC E545K 1,000 MYC * 100 100 80 750 80 BX795 GDC-0941 Mk2206 DMSO BX795 GDC-0941 Mk2206 DMSO BX795 GDC-0941 Mk2206 60 60 DMSO 500 MYC 40 40 p-AKT(S473) 250 20 20

Colonies in soft agar p-AKT(T308) Percentage of colonies (%) Percentage

Percentage of colonies (%) Percentage NS 0 0 0 AKT

MYC VectorPDK1 E545K PDK1 E545K DMSO BX795 shPTEN MK2206 GDC-0941

B EGMB-231 SUM159PT H1299 H460 Hep3B HepG2

HMEC RWPE-1 siNC siPDK#1 siPDK#2 siNC siPDK#1 siPDK#2 siNC si PDK1 siNC si PDK1 siNC si PDK1 siNC si PDK1 MYC sh PTEN E545K Vector PDK1 500 1,000 PDK1 PDK1 400 750 α MYC p110 300 500 Actin PTEN 200 250 MYC 100 H MB-231 Hs578T SUM159T T47D BT474 p-AKT(S473) Colonies in soft agar 0 0 BX795: –+ –+ –+ –+ –+

p-AKT(T308) VectorPDK1E545K VectorPDK1E545K MYC p-FOXO1 p-AKT(T308)

FOXO1 p-AKT(S473) F AKT p-FOXO3A HMEC RWPE-1 p-FOXO3A FOXO3A Actin E545K E545K PDK1 AKT Vector Vector PDK1 I PDK1 120 p-PKCδ(T505) p110α 100 PKCδ 80 MYC BT474 MYC- p-S6K(T229) 60 independent p-AKT(S473) T47D 40 p-ERK MDA-MB-231 p-AKT(T308) MYC-

Cell viability (%) 20 SUM159PT p-RSK2 Hs578T dependent AKT 0 p-SGK3 Actin 0 0.625 1.25 2.5 5.0 BX795 (μmol/L)

Figure 1. PDK1 induces cell transformation through MYC induction. A, soft-agar growth of HEK-TERV cells infected with retroviral constructs expressing empty vector, PDK1, MYC, shPTEN , or PIK3CA-E545K. B, immunoblot analysis of indicated proteins in HEK-TERV–derived cell lines. C, soft- agar growth of HEK-PDK1 and HEK-E545K cells transfected with nontargeting siRNA (siNC) or Myc siRNA, respectively. *, P < 0.01. D, soft-agar growth of HEK-PDK1, -E545K, and -MYC cells treated with BX795 (2.5 μmol/L), GDC-0941 (0.5 μmol/L), or MK2206 (0.5 μmol/L) for 14 days. Right, the changes in MYC and AKT after indicated drug treatments. E, soft-agar growth of HMEC and RWPE-1 cells expressing retroviral empty vector, PDK1, or E545K. F, immunoblot analysis of indicated proteins in HMEC and RWPE-1–derived cell lines. G, immunoblot analysis of indicated cancer cell lines treated with PDK1 siRNA. H, immunoblot analysis of indicated breast cancer cell lines treated with BX795 (2.5 μmol/L) for 24 hours. I, cell viability assay showing the dose response of a panel of breast cancer cell lines that are MYC-dependent (MDA-MB-231, SUM159PT, and Hs578T) and -independent (T47D and BT474) to BX795 treatment. All the data in the graph bars represent mean ± SEM; n = 3.

(sunitinib) showed a preferential inhibitory effect on the via- This ﬁ nding reveals a possible role of PLK1 in PDK1-induced bility of HEK-PDK1 cells as compared with the control cells transformation. Indeed, Western blot analysis showed an (Supplementary Fig. S2A). The two PLK1 inhibitors were fur- induction of PLK1 phosphorylation in all three PDK1-trans- ther validated in a secondary screen and thus were chosen for formed cell lines, but not in E545K- or MYC-transformed further study (data not shown). Further analyses in all three cells ( Fig. 2C ). Similar to the PDK1 inhibitor BX795, BI2536 epithelial systems showed that PDK1-transformed cells were treatment resulted in strong colony growth inhibition in much more sensitive to the PLK1 inhibitors compared with both PDK1- and MYC-transformed cells, but not in E545K- vector control or E545K-transformed cells (Fig. 2A and B). transformed cells ( Fig. 2D and Supplementary Fig. S2B).

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A BCD HEK HEK RWPE-1 HMEC 75 50 250 DMSO BI2536 GW843682X HEK RWPE-1 HMEC BI2536 120 40 200 100 50 30 Vector 80 PDK1 E545K MYC Vector PDK1 E545K Vector PDK1 E545K 150 20 25 p-PLK1(T210) 60 100 40 Vector 10

PDK1 Cell viability (%) 0 PLK1 50 20 E545k 0 Cell viability (%) 0 Actin Colonies in soft agar 0 000.63 1.25 2.5 5.010 0.1 0.5 1.0 2.5 VectorPDK1E545K VectorPDK1E545K PDK1 E545K MYC (nmol/L) (μmol/L) BI2536 10 nmol/L E FG HEK-PDK1 HEK-MYC HEK PDK1 MYC E545K BI2536 GW843682X BI2536 75 HEK RWPE-1 HMEC 50 30 50 50 40 40 II I 25

5 nmol/L 10 nmol/L 0.5 μ mol/L 1.0 μ mol/L 10 nmol/L 20 (%) 30 1 30 p-MYC Apoptosis (%) 0 20 20 MYC 10 siPLK1: –––#1 #2 #3 #1 #1 Sub-G 10 10 PLK1 p-PDK1 0 0 0 MYC PDK1 YC c-PARP M Vector PDK1 Vector PDK1 VectorPDK1 Actin E545K E545K E545K Actin BI2536 10 nmol/L HI HEK-PDK1 120 2,000 HEK-E545K

) 2,000 ) 3 DMSO 3 100 BI2536 1,500 1,500 80 MCF10A P < 0.01 HMEC MYC- 1,000 1,000 60 independent ** T47D 40 BT474 500 500

P < 0.001 Cell viability (%) MDA-MB-231 20 MYC- Tumor volume (mm volume Tumor 0 * (mm volume Tumor 0 Hs578T dependent 14710 0 SUM159PT 14710 0 0.63 1.25 2.5 5.0 10 Days of treatment Days of treatment BI2536 (nmol/L)

Figure 2. PLK1 is a crucial downstream effector of PDK1 for MYC activation and cell survival. A, cell viability of HEK-vector, HEK-PDK1, and HEK- E545K cells treated with the indicated concentrations of BI2536 and GW843682X for 4 days. B, cell viability of RWPE-1 and HMEC-derived cell lines treated with 10 nmol/L BI2536 for 4 days. C, immunoblot analysis of PLK1 in indicated cell lines. D, soft-agar growth of indicated cell lines treated with 10 nmol/L BI2536 for 14 days. E, immunoblot analysis in HEK-PDK1 and -MYC cells treated with BI2536 and GW843682X at indicated concentra- tion for 24 hours. F, apoptosis by sub-G1 analysis of indicated cell lines treated with 10 nmol/L BI2536 for 48 hours. G, apoptosis of indicated cell lines treated with NC or PLK1 siRNAs for 48 hours (top) and immunoblot analysis of indicated proteins (bottom). H, xenograft tumor growth of HEK-PDK1 and HEK-E545K cells in nude mice treated with 50 mg/kg BI2536 twice per week as described in Methods. Data are mean ± SEM (n = 5 for each group). I, cell viability assay showing the dose response of a panel of breast cancer cell lines that are MYC-dependent (MDA-MB-231, SUM159PT, and Hs578T) and -independent (T47D, BT474, MCF-10A, and HMEC) to BI2536 treatment.

Furthermore, like BX795, the PLK1 inhibitors BI2536 or tumors derived from HEK-PDK1 cells were highly sensitive GW843682X were able to eliminate endogenous MYC in to BI2536 treatment and displayed a strong tumor regres- HEK-PDK1 cells but also exogenous MYC in HEK-MYC sion following just two dosages, whereas the same treatment cells (Fig. 2E ). This fi nding suggests that exogenous MYC induced only tumor growth inhibition in E545K-derived is also sensitive to the perturbation of the basal level of xenograft tumors ( Fig. 2H ). PDK1–PLK1 signaling. Accordingly, in both HEK-PDK1 We further showed that the PLK1 regulation of MYC was and HEK-MYC cells, but not in HEK-E545K cells, BI2536 not limited to transformed cells but was also physiologi- treatment resulted in strong apoptosis, as shown by both cally relevant in human cancers, as either PLK1 knockdown fl uorescence-activated cell sorting (FACS) analysis ( Fig. 2F ) or BI2536 treatment resulted in endogenous MYC protein and increased caspase-3 activity (Supplementary Fig. S2C), depletion in various cancer cell lines without changing Myc whereas E545K-transformed cells mainly displayed G2 –M mRNA levels (Supplementary Fig. S3A–S3C). In addition, a arrest, a typical feature related to a mitotic effect following time-course analysis indicated that BI2536 treatment resulted PLK1 inhibition (Supplementary Fig. S2D). Furthermore, to in MYC depletion as early as 8 hours, concomitant with an confi rm the PLK1-specifi c effect of BI2536, PLK1 depletion early G2 –M arrest (Supplementary Fig S3D), indicating that by three independent siRNAs gave rise to similar effects on MYC downregulation is unlikely to be a result of the second- endogenous and exogenous MYC and apoptosis in PDK1- or ary effect of cell-cycle change. BI2536 treatment also resulted MYC-driven cells ( Fig. 2G ). These fi ndings suggest a crucial in more effective growth inhibition in MYC-dependent breast role for PDK1–PLK1 signaling in regulating MYC and can- cancer cell lines compared with MYC-independent cells cer cell survival. Consistent with the in vitro data, xenograft (except MDA-MB-231; Fig. 2I ). These results further support

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PDK1–PLK1–MYC Signaling in Transformation, Cancer Stem Cells, and Drug Resistance RESEARCH ARTICLE

ABC HCT116 MDA-MB-231 TT release: 4 8 12 h PDK1(+/+) PDK1(–/–) 4 shNC shPDK1 10 0.5% 2.9% shPDK1 6.0% shNC TT release: 04812 16 24 0 4 8 12 16 24 h TT release: 048048h

p-PDK1(S241) p-PDK1(S241) 0 10 PDK1 PDK1 4 10 1.1% p-PLK1(T210) 0.6% 0.1% p-PLK1(T210) PLK1

PLK1 0

MYC 10 Phosphor-H3 (S28) 0 1,000 001,000 1,000 p-Aurora A (T288) Actin DNA content (PI) Aurora A D E MYC 293T 1 100 200 603 PLK1 BX795 – – + – – p-AKT(T308) Active site BX912 – ––– + ATP binding site VX680 – + ––– p-AKT(S473) Substrate binding site Activation loop pCDNA-PDK1: – ++++ STKc AGC AKT PKC-like superfamily pCDNA-PLK1: ++++ + Exo-PLK1 PDK1 consensus motif p-PLK1(T210) p-FOXO3A 210 PLK1 T* LCGTPNY I APEVL Endo-PLK1 PKA 197T* LCGTPEYL A PEVL PLK1 p-S6K(T229) 308 AKT1 T* FCGTPEYL APEVL PLK1 IP: SGK1 256T* FCGTPEYL APEVL Exo-PLK1 p-S6K(T389) 320 p-PLK1(T210) SGK3 T* FCGTPEYL APEVL Endo-PLK1 S6K 229T* FCGT I EYMA P E IL PLK1 S6K PKCD 505T* FCGTPDY I A P E IL Input Actin PDK1

p-H3 (S10) FGIP: PLK1 BX795: – –––+ H3 BX795: ++– – – – BX912: – – – + – – – ––+ VX680: – – – – + + VX680: G1 (%): 53 292841 49 51 63 35 34 43 61 57 Recom-PDK1: – ++++ Recom-PDK1: – + – + – + Recom-PLK1: +++++ G2–M (%): 12 33 48 39 33 29 12 13 34 33 15 20 p-PLK1(T210) p-PLK1(T210) PDK1(+/+) PDK1(–/–) PLK1 PLK1 In vitro PDK1 In vitro PDK1 kinase assay 123456 kinase assay

Figure 3. PDK1 regulates PLK1 in vivo and in vitro. A, immunoblot analysis of indicated proteins in HCT116 PDK1 wild-type (PDK1 + /+) and knockout (PDK1 −/− ) cells. Cells were synchronized by double-thymidine block and released into cell cycle at indicated times. B, immunoblot analysis of indicated proteins in MDA-MB-231 shNC and PDK1 knockdown (shPDK1 ) cells. Cells were synchronized by double-thymidine block and released into cell cycle at indicated times. C, cells were synchronously released from double-thymidine arrest (TT) and harvested at the indicated times for FACS analysis. Percentages of cells positive for phosphor-H3 (S28) are indicated. PI, propidium iodide. D, PLK1 protein domain analysis (top) and PDK1 consensus motif alignment with other known PDK1 substrates (bottom). E, immunoblot analysis of immunoprecipitated PLK1 in 293T cells transfected with PLK1, or cotransfected with PDK1, with or without 2.5 μmol/L BX795, 5.0 μmol/L BX912, and 1.0 μmol/L VX680 treatment for 24 hours. F, in vitro immunoprecipitation-kinase assay using recombinant PDK1 and immunoprecipitated endogenous PLK1 from DLD1 cells as substrate. Cells were synchronized by a double-thymidine arrest and released in the presence or absence of 2.5 μmol/L BX795 or 1.0 μmol/L VX680 for 8 hours. PLK1 IP-kinase assay was conducted and the phosphorylation of PLK1 was assessed by using p-T210 PLK1 antibody. G, in vitro kinase assay using recombinant PDK1 and recombinant PLK1 with or without 1.0 μmol/L BX795, 1.0 μmol/L BX912, and 1.0 μmol/L VX680.

a role of PDK1–PLK1 signaling in supporting MYC-driven whereas in PDK1 −/− counterparts, we detected a much more tumorigenesis. defi cient PLK1 phosphorylation and MYC accumulation but not the phosphorylation of the PLK1-related kinase Aurora A PDK1 Induces PLK1 Phosphorylation in Human (refs. 20, 21 ; Fig. 3A ; Supplementary Fig. S4A). Cancer Cells We also probed the changes in the AKT–mTOR pathway We next sought to determine whether or not the PLK1 in these cellular contexts. Of note, compared with p-PLK1, activation by PDK1 seen in transformed cells represents a p-AKT (T308) was only modestly changed in this condition fi nding that is physiologically relevant in human cancer cells. in PDK1 −/− cells, whereas p-AKT (S473) and p-FOXO3A were To achieve this, we fi rst used the colon cancer HCT116 and even enhanced in both PDK1−/− cell lines (Fig. 3A ). This could DLD1 cells in which PDK1 is genetically knocked out (19 ). To be due to the inhibition of S6K in PDK1 −/− cells, leading to facilitate the detection of phosphorylation of PLK1, which is a a feedback activation of p-AKT (S473). In contrast, in a dif- mitotic kinase, cells were fi rst synchronized by double-thymi- ferent condition where cells were serum-starved and then dine block and then released into the cell cycle progressively stimulated with growth factors for early time points, we saw (Fig. 3A and Supplementary Fig. S4A). In PDK1 wild-type a clear p-AKT-(T308) inhibition in HCT116 PDK1 −/− cells cells, we noticed progressive induction of PDK1 phosphor- (Supplementary Fig. S4B). Thus, PDK1 regulates p-PLK1 ylation upon cell-cycle progression into mitosis as indicated and p-AKT (T308) in different growth conditions. To further by the elevated levels of phosphor-histone H3, which was consolidate the data, we also conducted PDK1 knockdown by accompanied by a similar pattern of PLK1 phosphorylation; shRNA in MDA-MB-231 cells. The result showed again that

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PDK1 knockdown resulted in ablation of PLK1 phosphoryla- well as in various cancer cell lines ( Fig. 4B and C ). We further tion and MYC accumulation (Fig. 3B ), as well as defi cient showed that PLK1 kinase activity is required for MYC protein entry into mitosis ( Fig. 3C ). These data consolidated a role accumulation, as the wild-type PLK1, but not the kinase-dead of PDK1 in driving PLK1 and MYC activation, not just in a mutant, induced strong MYC accumulation (Fig. 4D ). Cru- confi ned system but also in cancer cells. cially, in vitro kinase assays using either recombinant PLK1 Furthermore, in multiple cancer cell lines treated with ( Fig. 4E ) or endogenous PLK1 pulled down from the cancer BX795, GDC0941, or MK2206 upon double-thymidine block cells ( Fig. 4F ) showed a robust induction of S62 phosphor- and release (Supplementary Fig. S4C), we saw that BX795 ylation of recombinant MYC but not T58 phosphorylation, always blocked PLK1 phosphorylation and MYC accumu- which was reduced in the presence of BI2536. Importantly, lation, but inhibited AKT phosphorylation in a cell line– the endogenous PLK1 kinase activity toward MYC phospho- dependent manner (for example, AKT phosphorylation is rylation was strongly abolished in cells treated with BX795 not affected by BX795 in MDA-MB-231 cells). In contrast, (Fig. 4G ) or in PDK1 −/− cells (Fig. 4H ). Thus, these results not GDC0941 and MK2206 consistently inhibited AKT phos- only showed a direct phosphorylation of MYC by PLK1, but phorylation in each of these cell lines, but had little effect on also showed that PLK1 activity on MYC is crucially depend- PLK1 and MYC. Together, these data show the physiologic ent on PDK1. Together, these data reiterate the operation of relevance of AKT-independent PDK1–PLK1–MYC signaling PDK1–PLK1–MYC signaling in cancer cells. in cancer cells. We next investigated whether or not PLK1 is a potential PDK1–PLK1–MYC Signaling Drives Cancer- substrate of PDK1. PDK1 is known to regulate AGC kinases. Initiating Cell Maintenance and Self-Renewal Protein domain analysis indicated that the kinase domain During culturing of these transformed cells, we noticed of PLK1 is part of the AGC kinase family (Fig. 3D ). Inter- that HEK-PDK1 cells, and to a lesser extent HEK-MYC estingly, the amino acid sequence surrounding the Thr210 cells, displayed distinct morphologies from HEK-E545K contains a consensus motif for PDK1, which is found in cells and once they became confl uent in culture, started many known PDK1 substrates (Fig. 3D ), thus enhancing to form semiattached three-dimensional (3D) clusters on the possibility that PLK1 could be a potential substrate of the plate (Fig. 5A , top), suggesting that they displayed PDK1. Indeed, cotransfection of PDK1 and PLK1 into 293T tumorigenic and stem cell–like properties. This fea- cells, followed by PLK1 immunoprecipitation, showed that ture, however, was not observed in E545K-transformed PDK1 enhanced the phosphorylation of both the endog- cells (Fig. 5A ). Given the known role of MYC in induc- enous and exogenous PLK1, which was abolished when cells ing ESC- or CSC-like phenotypes in differentiated somatic were treated with BX795 or BX912, but not the Aurora A cells (16 ), it raises a possibility that PDK1, which acti- inhibitor VX680 (Fig. 3E ). This suggests that PDK1-induced vates MYC, may have a similar capacity in inducing CSC- PLK1 phosphorylation was unlikely to be an indirect effect like behavior. This hypothesis was fi rst tested using an of Aurora A, which might be coimmunoprecipitated with in vitro assay for spheroid formation in serum-free suspen- PLK1. Furthermore, in an in vitro kinase assay using endog- sion culture, a property associated with cancer stem/progen- enous PLK1 immunoprecipitated from DLD1 cells as a itor cells (22 ). We observed that PDK1- or MYC-transformed substrate, recombinant PDK1 added in the kinase assay HEK or HMEC cells formed large and abundant nonadher- induced PLK1 phosphorylation at T210, which was mark- ent tumorspheres after 7 days of growth in suspension cul- edly reduced in cells treated with BX795 (Fig. 3F ), indicating ture, whereas E545K-transformed cells were able to generate that the recombinant PDK1 can directly induce endogenous only a low number of small spheres (Fig. 5A , bottom and PLK1 phosphorylation in vitro. Importantly, in cells treated Supplementary Fig. S5A). These spheres were able to reform with VX680, where the PLK1 phosphorylation was greatly a monolayer when placed back to a tissue culture plate con- reduced as expected, recombinant PDK1 still boosted the taining serum-rich medium (Fig. 5B ). Furthermore, after PLK1 phosphorylation in the in vitro kinase assay (Fig. 3F ). dispersion into single cells, PDK1- or MYC-transformed This further excludes the possibility that PDK1 may induce HEK or HMEC cells reformed spheres with increasing PLK1 phosphorylation indirectly through Aurora A kinase. enrichments for at least four passages (Fig. 5C and Supple- Finally, in in vitro kinase assays using both PDK1 and PLK1 mentary Fig. S5B), indicating a gain of self-renewal capacity as recombinant proteins, we showed that PDK1 induced a that resembles a stem cell–like property. strong PLK1 phosphorylation, which was blocked by BX795 To show the tumor-initiating capacity of these transformed and BX912, but not by VX680 ( Fig. 3G ). Collectively, these cells in vivo , we next injected these cells at different numbers experiments provided evidence that PDK1 directly regulates into the fl anks of BALB/c nude mice. Strikingly, 1,000 HEK- PLK1 in human cancer cells. PDK1 cells were suffi cient to generate tumors in all six mice as early as 2 weeks (Fig. 5D , left). HEK-MYC cells seemed to PLK1 Directly Interacts with MYC and Induces be less tumorigenic and required 10,000 cells to generate a MYC Phosphorylation in a PDK1-Dependent similar size of tumors. In contrast, 10,000 HEK-E545K cells Manner were unable to induce tumors in the mice (Fig. 5D ). A further We next investigated whether PLK1 directly regulates MYC. experiment showed that as few as 100 PDK1 cells were suffi - Through coimmunoprecipitation (co-IP) assay, we showed an cient to give rise to xenograft tumors, whereas 3 × 10 6 E545K interaction between both exogenous PLK1 and MYC in 293T cells were required to generate observable tumors by 28 cells (Fig. 4A ). We also showed that the endogenous interac- days ( Fig. 5D , right). Importantly, PDK1-associated primary tion between the two proteins occurs in HEK-PDK1 cells as xenograft tumors were self-renewable, as determined by the

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PDK1–PLK1–MYC Signaling in Transformation, Cancer Stem Cells, and Drug Resistance RESEARCH ARTICLE

A BC IP: IgG PLK1 MYC Input

293T HEK-TERV IP: PLK1 IP: MYC Input IP: PLK1 Input PLK1: ++– +++––+ SW480 HCT116 MDA-MB-231 SW480 HCT116 MDA-MB-231 SW480 HCT116 MDA-MB-231 SW480 HCT116 MDA-MB-231 MYC: – ++ – ++ – ++ Vector PDK1 Vector PDK1 MYC MYC MYC

PLK1 PLK1 PLK1

D EFDLD1 HCT116 BI2536: – – + 293T PLK1: – + + IP : PLK1: Vector WT KD MBP-MYC: ++ + lgG PLK1 PLK1 WCL lgG PLK1 BI2536: ––+ – –– MYC: –––+++ p-MYC(S62) MBP-MYC: +++ – + + MYC p-MYC(T58) p-MYC(S62) PLK1 MYC MYC In vitro In vitro

Actin kinase assay PLK1 PLK1 kinase assay

GHlgGIP: PLK1 IP: lgG PLK1 +/+ –/– +/+ –/– BX795: ––++ MBP-MYC: ++++ MBP-MYC: + PDK1 + PDK1 + PDK1 + PDK1 p-MYC(S62) p-MYC(S62) MYC MYC p-PLK1(T210)

In vitro p-PLK1(T210) In vitro

kinase assay PLK1

Figure 4. PLK1 interacts with MYC and induces MYC phosphorylation. A, co-IP analysis in 293T cells transfected with ectopic PLK1, MYC, or both. B, co-IP analysis of endogenous PLK1 and MYC in HEK-Vector and HEK-PDK1 cells. C, co-IP analysis of endogenous PLK1 and MYC in cancer cell lines. D, immunoblot analysis of MYC protein expression in 293T cells transfected with empty vector, PLK1 wild-type (WT) or kinase-dead mutant of PLK1 (KD) in the absence or presence of ectopic MYC. E, immnoblot analysis of in vitro kinase assay using recombinant PLK1 and recombinant MYC proteins in the presence or absence of BI2536. Phosphorylation of MYC was assessed by indicated MYC antibodies. F, immnoblot analysis of in vitro kinase assay using immunoprecipitated PLK1 and recombinant MYC proteins in the presence or absence of BI2536. G, immnoblot analysis of in vitro kinase assay using immunoprecipitated PLK1 from DLD1 cells treated with or without 2.5 μmol/L BX795. H, immnoblot analysis of in vitro kinase assay using immunoprecipitated PLK1 from DLD1 and DLD1 PDK1 −/− cells. IgG, immunoglobulin G. ability to form secondary and tertiary tumors using as few as MEF-PDK1 cells formed colonies resembling the ESC-like 100 xenograft tumor cells (Fig. 5E ). These in vitro and in vivo morphology and were alkaline phosphatase-positive (Sup- data showed a strong tumorigenicity of PDK1-transformed plementary Fig. S5G), though we found that these colonies cells with self-renewal capacity. were unable to maintain the ESC-like morphology in the We also tested the ability of PDK1 in inducing mouse subsequent passages, probably due to an incomplete repro- embryonic fi broblast (MEF) reprogramming. To achieve this, gramming. Thus, in both human epithelial cells and MEFs, we used p53-defi cient MEFs, as immortalization by p53 inac- PDK1 is able to induce PLK1 and MYC activation and ESC- tivation has been shown to enhance MEF reprogramming like properties. effi ciency ( 23, 24 ). Again, PDK1 but not E545K was also able Aberrant high PDK1 activity has been shown in inva- to induce MYC activation, as well as tumorsphere formation sive and metastatic breast tumor samples ( 25 ). To show in immortalized Trp53 −/− MEFs (Supplementary Fig. S5C and the capacity of the PDK1–PLK1–MYC pathway in regulat- S5D). In the PDK1 sphere populations, we detected strongly ing CSCs, we used the highly invasive breast cancer MDA- increased expression of embryonic stem pluripotency fac- MB-231 and SUM159PT cells that contain a high percentage tors SOX2 and OCT4 as assessed by both quantitative PCR of CD44+ /CD24 − /low CSC-like cells (26 ). Intriguingly, PDK1– (qPCR) and confocal imaging compared with the monolayer PLK1–MYC signaling was found to be enriched in CD44+ / growth (Supplementary Fig. S5E and S5F). When these MEF- CD24 − /low cells compared with the non-CD44 + /CD24− /low cells PDK1 cells were cultured in ESC medium containing the ( Fig. 5F ). Knockdown of PDK1/PLK1 or treatment with their differentiation inhibitor LIF (leukemia inhibitory factor), corresponding inhibitors BX795/BI2536 resulted in marked

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A BC Vector PDK1 MYC E545K Monolayer 20 HEK-PDK1 HEK-MYC 15 PDK1 DMEM 10

umorsphere (%) 5 T MYC Sphere 0 01234 Sphere passages

D EF MDA- 100 cells PDK1 1 × 104 (6/6) MB-231 SUM159PT 1,000 500 cells × 4 500 PDK1 100 (3/6) MYC 1 10 (6/6) 9/9 9/9 5/5 5/5 MYC 100 (1/6) 100 /high/ /high /high/ /high/ /high /high/ 3 /low /low ) ×

PDK1 1 10 (6/6) + + + – + + + – 3 400 E545K 3 × 106 (6/6) 750 MYC 1 × 103 (3/6) 7/9 CD44 CD24 CD44 75 CD24 CD44 CD24 CD44 CD24 E545K 1 × 104 (0/6) 300 p-PDK1(S241) 500 50 3/6 * 200 PDK1 25 p-PLK1(T210) 250 100

Tumor incidence (%) Tumor PLK1 Tumor volume (mm volume Tumor ** 0 0 0 1st 2nd 3rd MYC 11 14 17 20 23 14 17 20 23 26 29 Passages Actin (Days) (Days)

GH I J MDA-MB-231

4 MDA-MB-231 MDA-MB-231 20 MDA-MB-231

10 NC 20 750 3 10 cells cells 2 15

10 15 –/low 1 –/low 500 10 CD24-APC 0 16.7% 10 10 0 1 2 3 4 10 /CD24 /CD24 10 10 10 10 10 + * * + 4

10 * PDK1KD * 250

3 * 5 * 5

10 ** ** * * 2 No. of mammospheres No. 10 % of CD44 1 % of CD44 0 0 0 10 CD24-APC 0 3.7% NC NC 10 100 101 102 103 104 DMSOBX795BX912BI2536 DMSOBX795BI2536 MK2206 PDK1 KD CD44-FITC PDK1PLK1 KDPLK1 KD1 KD2 GDC-0941 PLK1 KD1

Figure 5. PDK1–PLK1–MYC signaling drives CSC-like phenotypes. A, representative phase-contrast images of HEK-vector, -PDK1, -MYC, or -E545K cells grown in monolayer culture (top). Bottom, tumorsphere formation in suspension culture without serum. Scale bar represents 100 μm. B, spheres formed in suspension culture reattached when transferred back to gelatin-coated culture plates in Dulbecco’s Modifi ed Eagle Medium (DMEM), 10% FBS, and the sphere reformed a monolayer for 48 hours. Scale bar represents 100 μm. C, self-renewal capacity of PDK1- and MYC-transformed cells. Primary tumorspheres were trypsinized into single cells and reformed spheres 7 days later for four passages. D, xenograft tumor growth in nude mice. The indicated number of HEK-PDK1, -MYC, or -E545K cells were injected. Data are mean ± SEM. *, P < 0.01; **, P < 0.005. E, xenograft tumor formation frequencies of tumor-initiating cells derived from the fi rst, second, and third passage tumors arising from HEK-PDK1 cells. F, immunoblot analysis showing the PDK1–PLK1–MYC signaling in CD44+ /CD24 −/ low or non-CD44 +/CD24 − /low populations. G, representative FACS profi les for CD44+ /CD24 − /low or non-CD44 +/CD24 −/ low populations in MDA-MB-231 and MDA-MB-231-PDK1 KD cells. Inset: isotype control. H, bar graphs showing the percentages of CD44+ /CD24 −/ low cells in MDA-MB-231 cells depleted of PDK1 or PLK1. *, P < 0.005. I, bar graphs showing the percentages of CD44+ /CD24 − /low cells in MDA-MB-231 cells treated with indicated inhibitors. *, P < 0.01; **, P < 0.005. J, bar graphs showing the number of tumorspheres of MDA-MB-231 cells depleted of PDK1/PLK1 (left) or treated with BX795/BI2536. *, P < 0.01. Data are mean ± SEM (n = 3).

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PDK1–PLK1–MYC Signaling in Transformation, Cancer Stem Cells, and Drug Resistance RESEARCH ARTICLE

reduction of the CD44 +/CD24 − /low population ( Fig. 5G–I ). a marked upregulation of miR-17-92 and downregulation In contrast, PI3K/AKT inhibitors GDC-0941 and MK-2206 of let-7 s in PDK1 and MYC cells, but not in E545K cells were unable to do so ( Fig. 5I ). Corresponding to the reduced (Fig. 6F ). Because let-7 suppresses its own negative regulator CD44 +/CD24 − /low cells, PDK1 or PLK1 inhibition either by LIN28B (33 ), it is likely that PDK1 enforces a feedback loop gene knockdown or inhibitor treatment resulted in marked via MYC-LIN28B –mediated let-7 downregulation to support inhibition of tumorsphere formation in MDA-MB-231 cells the self-renewal program. Finally, we showed that BI2536 (Fig. 5J ). treatment of HEK-PDK1 cells resulted in reduced expression of some ESC- or CSC-related genes, including EPCAM, SOX2, SALL4 , and JAG2 (Fig. 6G ), validating a role of PLK1 PDK1 Activates Embryonic Stem in the PDK1-mediated CSC gene signature. These fi ndings or CSC-Like Transcriptional Programs indicate that PDK1 is able to evoke multiple transcriptional It is known that MYC is able to activate ESC-like tran- programs that coordinately induced a remarkable repro- scriptional programs in adult epithelial cells, resulting in a gramming toward a state resembling CSCs. In this process, CSC-like phenotype in the appropriate genetic context (16 ). MYC is one important factor but not the only one that To characterize the transcriptional program underlying the modulates the reprogramming. PDK1-induced CSC-like behavior, we compared the gene expression profi les in HEK-PDK1, -MYC, or -E545K cells. PDK1-Induced CSC-Like Gene Signature Is Signifi cant analysis of microarray identifi ed 1,750, 1,080, Relevant to Human Cancers and Is Associated and 297 differentially expressed genes in these transformed with Aggressive Tumor Behavior cells when compared with nontransformed control cells, Aberrant gene expression associated with embryonic stem respectively (false discovery rate < 0.05; P < 0.01; Supple- cell identity, including ESC genes, MYC targets, and Poly- mentary Tables S1–S3). Gene Venn Diagram analysis shows comb targets, has been found in poorly differentiated tumors that HEK-PDK1 cells shared a robust transcriptional pro- (14–16 ). By further interrogating several previously published gram with HEK-MYC cells, but had little overlap with HEK- datasets collectively, we showed that the above ESC-related E545K cells (Fig. 6A ). In addition to the PDK1 and MYC genes were signifi cantly enriched in the PDK1-dependent common gene set, PDK1 also regulates a unique set of 784 transcriptome, including upregulation of 97 ESC-expressed genes. We further stratifi ed the PDK1- or MYC-regulated genes and downregulaton of 182 Polyccomb targets (PRC genes into 889 upregulated and 1,151 downregulated genes genes; Supplementary Fig. S6A and Supplementary Table S5). via gene cluster analysis (Fig. 6B ). Notably, a number of In contrast, the E545K-associated transcriptional program well-known genes implicated in ESC pluripotency or main- displayed a distinct gene set that is not signifi cantly associ- tenance, including SOX2, LIN28B , SALL4 , and EZH2 (27 ), or ated with ESCs (Supplementary Fig. S6A). CSCs, including EPCAM , ALDH1A , and S100A4 (28 ), were We next determined whether PDK1-driven ESC-like gene upregulated in both HEK-PDK1 and HEK-MYC cells, but expression is of clinical relevance to human malignancy. not in HEK-E454K cells (Fig. 6B ). JAG2, which was recently Gene set enrichment analysis (GSEA; ref. 35 ) of several previ- shown to be a MYC target (29 ) with a role in modulating ously published datasets showed that PDK1-induced ESC- CSCs, was also markedly induced by PDK1 and MYC but like genes were found to be signifi cantly enriched in colon not by E545K. In addition, a set of genes encoding secreted and lung tumor samples as compared with the normal con- inhibitors of autocrine signaling, including DKK1, SFRP1 , trols, whereas the Polycomb targets were inversely correlated and BMP4 , whose reduction has been recently shown by in these samples (Supplementary Fig. S6B). Moreover, in Scheel and colleagues (30 ) to enable self-renewal of epithe- breast cancer, deregulation of these genes was signifi cantly lial cells, were strongly repressed in PDK1 and MYC cells but correlated with the high-grade tumors as compared with not in E545K cells. CD24, a negative selection marker for the low-grade tumors (Supplementary Fig. S6C). This indi- CSCs (31 ), was also selectively repressed in PDK1 and MYC cates that aberrant expression of these PDK1-regulated ESC cells. The array results of selected genes were further vali- genes is associated with malignant progression from normal dated by quantitative real-time PCR ( qRT-PCR; Fig. 6C ) and to aggressive tumors. In addition, we also showed that the Western blotting (Fig. 6D ). Notably, many genes coregulated PDK1-regulated ESC-like gene signature was associated with by PDK1 and MYC, including SOX2 , EPCAM , JAG2 , and poor disease outcome as shown in the survival analysis of S100A4 , were more affected by PDK1 than MYC. In total, breast and lung cancer cohorts (Supplementary Fig. S6D), we identifi ed 668 genes showing such a pattern (Fig. 6E and providing a prognostic value of these genes. Together, these Supplementary Table S4), which is consistent with a more fi ndings show that the PDK1-activated ESC-like gene signa- robust role of PDK1 than MYC in tumorigenesis. ture we identifi ed from the in vitro culture system is clinically We also investigated the changes of microRNAs (miRNA, relevant to human cancers arising in distinct tissues and miR) that are MYC-associated and implicated in ESC self- support a link of PDK1–MYC signaling to aggressive cancer renewal. LIN28B is a known MYC target and is able to behavior. inhibit the biogenesis of the let-7 family miRNAs (32 ). Inhibition of let-7 miRNAs has been shown to enhance BI2536 Synergizes with PI3K–mTOR Inhibitor reprogramming of somatic cells to induced pluripotent BEZ235 to Induce Robust Apoptosis and Tumor stem (iPS) cells (33 ). MYC also transactivates the miR-17-92 Growth Inhibition in Colorectal Cancer cluster, which is also implicated in ESC maintenance (34 ). We have previously shown that mTOR inhibition by Consistent with MYC and LIN28B elevation, we detected rapamycin or mTOR/Raptor knockdown induces MYC

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A BE PDK1 MYC Vector E545K 6 PDK1 CD24 DKK1 (1,750) MYC 4 SFRP1 ) n = 318, (1,080) 2 BMP4 P = 7.97e–12

1,151 2 LIN28B 784 821 138 EPCAM 0 JAG2 S100A4 –2 105 16 ALDH1A2 change (log Fold SALL4 n = 350, 40 E545K 889 –4 FOXA2 P < 2.2e–16 136 (297) MYB SOX2 PDK1 MYC MYC PDK1

C DF PDK1 MYC E545K BMP4 DKK1 PDK1 MYC E545K PDK1 MYC E545K SFRP1 Vector CD24 SOX2 let-7e S100A4 EPCAM let-7b MYB FOXA2 let-7i FOXA2 ALDH1A2 LIN28B miR-92a JAG2 JAG2 miR-20a EPCAM MYB miR-18a SALL4 miR-17 LIN28B SALL4 SOX2 S100A4 –3 0 3 –3 0 3 –3 0 3 Fold change –5 00555–5 –5 0 CD24 (log2) Fold change Actin (log2)

1.00 1.5 1.00 1.00

0.75 0.75 0.75 1.0

0.50 0.50 0.50 0.5 0.25 0.25 0.25 Relative expression level expression Relative Relative expression level expression Relative Relative expression level expression Relative Relative expression level expression Relative 0.00 0.0 0.00 0.00 0 24 48 72 h 0 24 48 72 h 0 24 48 72 h 0 24 48 72 h

BI2536 (10 nmol/L)

Figure 6. PDK1 evokes ESC-like gene expression proﬁ le. A, Venn diagram showing the overlapping of differentially expressed genes in HEK-PDK1, -MYC, or -E545K as compared with HEK-vector control cells. B, heatmap of differentially expressed genes in HEK-PDK1, -MYC, or -E545K cells. C, qRT-

PCR analysis of representative genes in HEK-transformed cells. Data are shown as gene expression fold change (log2 ) relative to HEK-vector cells. Red < and green bars indicate upregulation and downregulation, respectively. Black bars indicate 0.6-fold change in log 2 (1.5-fold in linear scale). Data are mean ± SEM; n = 3. D, immunoblot analysis of indicated proteins. E, 318 upregulated and 350 downregulated genes show signiﬁ cant differences between PDK1 and MYC regulation. Average gene expression levels indicating a higher impact of PDK1 on these genes. F, qRT-PCR analysis of indicated miRNAs in HEK-PDK1, -MYC, and -E545K cells. Data are presented as in C. G, qRT-PCR analysis of indicated genes in HEK-PDK1 cells treated with 10 nmol/L BI2536 at indicated times. Data are means ± SEM (n = 3).

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accumulation in colorectal cancer, which can be inhibited nation of BI2536 and PI3K–mTOR dual inhibitors such as by PDK1 inhibition, resulting in rapamycin sensitization ( 5 ). BEZ235 may represent a promising treatment strategy for As our data now indicate that PLK1 is required for PDK1– colorectal cancer. MYC signaling, together with our further observation that PLK1 is highly expressed in colorectal cancer tumors compared with adjunct normal regions (Supplementary Fig. S7A DISCUSSION and S7B), we hypothesized that the PLK1 inhibitor could PDK1 Regulation of PLK1–MYC Signaling also sensitize colorectal cancer cells to mTOR inhibitors in Human Cancer Cells through abolishing mTOR inhibitor–induced MYC activa- Although PI3K–AKT signaling has been considered to tion. Classical mTOR inhibitors such as rapalogs are known be the main signaling pathway associated with PDK1 in to induce compensatory feedback activation of PI3K–AKT oncogenesis, our study uncovers another arm of signaling due to S6K inhibition. BEZ235, a dual PI3K–mTOR kinase that routes to PLK1–MYC to confer malignant phenotypes. inhibitor, is able to overcome the feedback AKT activation Importantly, the pathway we identifi ed using a chemical and is currently being tested in several clinical trials either genetic approach with a PDK1-transformed cell line has as a single agent or in combination with other therapeutics. been validated to be relevant in human cancers, as shown in Unlike rapamycin treatment, which induced both AKT and multiple cancer cell lines derived from various tissue types. MYC activation in colorectal cancer cells, BEZ235 did not Although in our system we detected AKT phosphorylation induce AKT activation but retained the ability to induce at T308 by PDK1, we did not see AKT phosphorylation at MYC (Fig. 7A ). Of note, neither drug induced ERK activa- S473 that is required for a full AKT activation (6 ). This is in tion in colorectal cancer, which is, however, often seen contrast to PI3K-transformed cells in which both AKT phos- in breast cancer cells (36 ). As expected, BI2536 cotreat- phorylations are strongly induced. This observation is con- ment effectively removed BEZ235-induced MYC induction sistent with a recent report showing that PDK1-defi ciency in ( Fig. 7B ). In these cells, BI2536 or BEZ235 alone failed colon cancer cells has only a modest effect on p-AKT (T308) to induce signifi cant apoptosis, but their combination, and has no effect on p-AKT (S473; ref. 19 ). Our data thus which resulted in inhibition of both MYC and p-4EBP1, indicate that PDK1 signaling might be wired differentially in induced massive apoptosis, as evidenced by strong detection certain oncogenic contexts to confer a growth advantage that of PARP cleavage (Fig. 7B ), cells in sub-G (Fig. 7C), and 1 becomes less dependent on AKT. Indeed, previous reports caspase-3 activation (Supplementary Fig. S7C). The com- have shown that PDK1 is not linked to PI3K signaling in a binatorial effect was synergistic, as shown by combination PTEN-defi cient tumor model (8 ) and can route through the index analysis (Supplementary Fig. S7D) and further con- AKT-independent pathway for cell survival in some cancer fi rmed by time-course analysis of cell viability ( Fig. 7D ) and cell lines (10 ). As PDK1 has attracted much attention as a long-term colony formation assay (Supplementary Fig. S7E). potential therapeutic target in cancer, we propose that MYC Finally, to assess the potential of the combination strategy can be an alternative pharmacodynamic marker for the evalu- in vivo , SW480 and HT15 cells were injected subcutane- ation of small-molecule PDK1 inhibitors under preclinical ously into nude mice to establish tumor xenografts. We and clinical development. showed that BEZ235 also induced MYC accumulation in the xenograft tumors, which can be inhibited via combination with BI2536 (Supplementary Fig. S7F). Accordingly, the Therapeutic Targeting of PDK1–PLK1 Signaling combination treatment induced synergistic tumor growth in MYC-Dependent Tumors inhibition compared with the single-agent treatment, vali- An intriguing fi nding of this study is the identifi cation dating the in vitro fi ndings ( Fig. 7E ). of the crucial role of PDK1–PLK1–MYC signaling for can- Like BEZ235, a specifi c mTOR inhibitor, PP242 ( 37 ), also cer cell survival. We provide evidence that PDK1 induces generated similar results on MYC, p-4EBP1, and apoptosis PLK1 phosphorylation and PLK1 binds to and induces MYC when combined with BI2536 (Supplementary Fig. S8A and phosphorylation and protein accumulation widely in cancer S8B). In contrast, BI2536, though it also blocked rapamycin- cells. A previous report has shown that PLK1-induced MYC induced Myc accumulation (Supplementary Fig. S8C), did phosphorylation is required for SCFβ TrCP-mediated MYC pro- not enhance apoptosis (Supplementary Fig. S8D), but only tein stabilization during late-stage cell-cycle progression ( 40 ). potentiated the antiproliferation effect and xenograft tumor We further show here that PLK1 can directly bind to MYC. growth inhibition (Supplementary Fig. S8E and S8F). This is Regardless of whether or not PDK1–PLK1 signaling regulates probably due to the inability of rapamycin to block 4EBP1 MYC stability through a similar or distinct mechanism, the phosphorylation (Supplementary Fig. S8C) as previously direct regulation of MYC by PDK1–PLK1 signaling immedi- shown. 4EBP1, but not S6K, has been recently shown to be ately suggested a therapeutic approach targeting MYC-driven the key effector of the mTOR pathway responsible for cell tumors. Indeed, our data show a preferential killing of small- proliferation and survival ( 38 ), and additional inhibition of molecule inhibitors of PDK1 or PLK1 in MYC-dependent 4EBP1 phosphorylation seems to be required for apoptosis breast cancer cells compared with MYC-independent breast induction in response to the AKT inhibitor (39 ). Thus, the cancer cells. Given that a clinical inhibitor of MYC is not simultaneous inhibition of both MYC and 4EBP1 phosphor- available, small-molecule inhibitors such as BI2536, which is ylation upon combination of BI2536 and BEZ235 seemed to currently in late-stage clinical trials, may provide an alterna- be crucial for apoptosis induction of colorectal cancer cells. tive anti-MYC strategy. Given that PLK1 is often found to be Taking all the data together, we conclude that the combi- overexpressed in human cancers (41 ), therapeutic targeting of

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A B DLD1 SW480 HT15 DLD1 BI2536: −−+ +++ −− −− ++ BEZ235: −−++ − − ++−− ++ Rapa BEZ235 p-MYC(S62) −+−+ MYC MYC p-AKT(S473) p-S6K(T389) p-4EBP1(T36/47) p-AKT(S473) p-4EBP1(T70)

AKT 4EBP1

p-ERK c-PARP

Actin p-ERK Actin

CD E DLD1 SW480 DLD1 50 750 DMSO 1,500 Vehicle BI2536 BI2536 40

) BEZ235

BEZ235 3 1,250 500 BI + BEZ (%) 30 BI + BEZ 1 1,000 20 250 750 Sub-G 10 RLU ( × 1,000) 500 0 0

40 SW480 (mm volume Tumor 250 750 SW480 0 30 1471013 16

(%) 500 (Days of posttreatment) 1 20

Sub-G 10 250 HT15 RLU ( × 1,000) Vehicle 0 4,000 0 BI2536

) BEZ235 60 HT15 3 + 1,500 HT15 3,000 BI BEZ 50

40 2,000 (%) 1,000 1 30 20 1,000

Sub-G 500 Tumor volume (mm volume Tumor 10 RLU ( × 1,000) 0 0 0 1471013 16 1234 (Days of posttreatment) DMSOBI2536 (Days) BEZ235BI + BEZ

Figure 7. BI2356 synergizes with BEZ235 to induce synthetic lethality in colorectal cancer both in vitro and in vivo. A, immunoblot analysis of DLD1 cells treated with 100 nmol/L rapamycin or 100 nmol/L BEZ235 for 48 hours. B, immunoblot analysis of DLD1, SW480, and HT15 cells treated with

10 nmol/L BI2536 alone, 100 nmol/L BEZ235 alone, or the combination for 48 hours. C, sub-G 1 detection of apoptosis in DLD1, SW480, and HT15 cells treated as in B. D, the growth curves of DLD1, SW480, and HT15 cells treated with 10 nmol/L BI2536 alone, 100 nmol/L BEZ235 alone, or the combination for 4 days. RLU, relative luminescence units. E, xenograft tumor growth of SW480 and HT15 cells in nude mice treated with BI2536 at 50 mg/kg, BEZ235 at 35 mg/kg, or both, every other day as described in Methods. Error bars represent ± SEM ( n = 6 per group). Data are mean ± SEM (n = 3).

OF13 | CANCER DISCOVERYOCTOBER 2013 www.aacrjournals.org

PDK1–PLK1–MYC Signaling in Transformation, Cancer Stem Cells, and Drug Resistance RESEARCH ARTICLE

PLK1 may yield a more favorable therapeutic index in MYC- study were obtained from Axon Medchem. Information regarding associated tumors. plasmid DNA vectors and stable cell line construction is provided in the Supplementary Materials and Methods. Role of PDK1–PLK1–MYC Signaling in Driving Tumor-Initiating Cells Cell Cultures The main characteristic of the PDK1-induced transforma- Cell cultures and various cellular assays are described in the tion is that it is able to induce both the genotype and pheno- Supplementary Materials and Methods. All cancer cell lines were type of CSCs that have been proposed to account for tumor purchased from American Type Culture Collection, and no authenti- cation of cell lines was done by the authors. initiation, progression, and chemoresistance ( 13 , 31 ). We show that as few as 500 PDK1-transformed cells can induce robust Mouse Experiments tumorigenicity and that PDK1 activates clinically relevant transcriptional programs associated with poor disease outcome. In All the experiments in xenografts are described in the Supplemen- tary Materials and Methods. addition, PDK1 or PLK1 inhibition also resulted in disrup- tion of both embryonic and adult stem cell self-renewal while Immunobloting, Immunoprecipitation, and In Vitro inducing differentiation. Activation of an ESC-like signature Kinase Assays and an ESC-like phenotype in differentiated somatic cells also Details are described in the Supplementary Materials and Methods. indicates that the embryonic stem program can be reactivated during the course of tumor progression and is not necessarily Gene Expression, Data Analysis, and RT-PCR Analysis inherited from a stem cell-of-origin. This notion is consistent The microarray hybridization was conducted using the Illumina with a recent report from Chaffer and colleagues ( 42 ) showing Gene Expression Sentrix BeadChip HumanHT-12_V4 (Illumina), spontaneous CSC generation from mammary epithelial cells. and the data were analyzed using the GeneSpring GX 11.0.2 (Agilent Furthermore, consistent with a previous report that PDK1 Technologies). Detailed information can be found in the Supplemen- is hyperactivated in invasive and metastatic breast cancer tary Materials and Methods. Primers used in RT-PCR analysis are (25 ), we show that PDK1 or PLK1 inhibition in highly inva- described in Supplementary Table S6. sive breast cancer MDA-MB-231 cells resulted in depletion of CSC-like CD44 +/CD24 − /low populations and accordingly Statistical Analysis strongly reduced tumorsphere formation, whereas PI3K– PDK1-regulated ESC-like and PRC gene signature defi nition is AKT inhibition did not have such effects. Thus, small- described in the Supplementary Experimental Procedures. GSEA ( 35 ) molecule inhibition of PDK1–PLK1–MYC signaling for was conducted to assess the degree of correlation between PDK1- elimination of CSCs may provide a targeted therapy to regulated gene signatures and cancer phenotypes on different human overcome recurrence of aggressive breast tumors following patients. Survival curves were calculated using the Kaplan–Meier sur- chemotherapy. vival analyses and the quantiles-rank test. Detailed statistical analysis is included in the Supplementary Data. Data are presented as mean Combination of PLK Inhibitor and PI3K–mTOR ± SEM, unless otherwise stated. A Student t test was used to compare Inhibitor for Colorectal Cancer two groups for statistical signifi cance analysis. An additional therapeutic application of our studies is the Accession Number identifi cation of strategies to overcome resistance to mTOR- targeted therapy in colorectal cancer. Drug resistance and The microarray data are deposited into the Gene Expression Omnibus with the accession number GSE30669. tumor recurrence is the main cause of patient relapse, possibly owing to recurrence of CSCs. We have previously discovered Disclosure of Potential Confl icts of Interest that mTOR inhibition induces MYC activation, a compensa- No potential confl icts of interest were disclosed. tory effect mitigating the antiproliferative effect of mTOR inhibitors in colorectal cancer ( 5 ). We now show that PLK1 Authors’ Contributions inhibitor blocks mTOR inhibitor–induced MYC activation, Conception and design: J. Tan, Q. Yu providing a rational approach to developing a new combina- Development of methodology: J. Tan, P.L. Lee, P. Guan, M. Feng tion therapy for colorectal cancer. Specifi cally, a low dose Acquisition of data (provided animals, acquired and managed of PLK1 inhibitor BI2536 plus PI3K–mTOR dual inhibitor patients, provided facilities, etc.): Z. Li, P. Guan, S.T. Lee, Z.N. Wee, BEZ235 induced massive apoptosis in colorectal cancer cells Y.C. Lim, Q. Yu and a synergistic loss of colony formation, suggesting that this Analysis and interpretation of data (e.g., statistical analysis, strategy might be useful in colorectal cancer. Given that both biostatistics, computational analysis): J. Tan, P. Guan, M. Feng, drugs are in late-stage clinical trials, we hope that our fi ndings C.Z. Lim, Y.C. Lim, R.K.M. Karuturi will spur clinical trials in colorectal cancer for the combination Writing, review, and/or revision of the manuscript: J. Tan, Q. Yu Administrative, technical, or material support (i.e., reporting of BEZ235 and BI2536 to improve the therapeutic outcome. or organizing data, constructing databases): P.L. Lee, P. Guan, M.Y. Aau, S.T. Lee, C.Z. Lim, E.Y.J. Lee, Z.N. Wee METHODS Study supervision: Q . Y u Constructs and Reagents Acknowledgments Human full-length PDK1, MYC, PIK3CA-E545K, and PLK1 were The authors thank Dr. William C. Hahn for the HEK-TERV cells. cloned into PMN–IRES–GFP retroviral vector and introduced into The authors also thank Dr. Fu Zheng for the human PLK1 plasmids human epithelial cells and MEFs. All kinase inhibitors used in this and Dr. Luca Primo for the PDK1 kinase-dead mutant construct.

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RESEARCH ARTICLE Tan et al.

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PDK1–PLK1–MYC Signaling in Transformation, Cancer Stem Cells, and Drug Resistance RESEARCH ARTICLE

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

Jing Tan, Zhimei Li, Puay Leng Lee, et al.

Cancer Discovery Published OnlineFirst July 25, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-12-0595

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