PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumor-initiating cell...

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PDK1 Signaling Towards PLK1-Myc Activation Confers Oncogenic Transformation and Tumor Initiating Cell Activation and Resistance to mTOR-targeted Therapy Jing Tan1, Zhimei Li1, Puay Leng Lee1, Peiyong Guan2, Mei Yee Aau1, Shuet Theng Lee1, Min Feng1, Cheryl Zihui Lim1, Eric Yong Jing Lee1, Zhen Ning Wee1,3 Yaw Chyn Lim4, R. K. Murthy Karuturi2 and Qiang Yu1, 4, 5,*

1 Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore. 2 Information and Mathematical Science, Genome Institute of Singapore, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore.

3Graduate School for Integrative Sciences and Engineering, National University of Singapore

4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore 5Cancer and Stem Cell Biology, DUKE-NUS Graduate Medical School of Singapore Running Title: PDK1- PLK1-Myc signaling in transformation, cancer stem cells and drug resistance. *Correspondence: [email protected] Qiang Yu, M.D. Ph.D. Cancer Therapeutics and Stratified Oncology Genome Institute of Singapore Email: [email protected] Fax: 65-6808-9003 Disclosure of Potential Conflicts of Interest No potential conflicts of interests were disclosed.

Research. on June 8, 2016. © 2013 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 25, 2013; DOI: 10.1158/2159-8290.CD-12-0595

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ABSTRACT

Although 3-Phosphoinositide–dependent protein kinase-1 (PDK1) has been

predominately linked to 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 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 PLK1 inhibitor synergizes with

mTOR inhibitor to induce synergistic anti-tumor effect in colorectal cancer by

antagonizing a compensatory Myc induction. These findings 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 identifies PDK1-PLK1-Myc signaling as a new oncogenic

pathway driving oncogenic transformation and cancer stem cell self-renewal. Targeted

inhibition of PDK1/PLK1 is robust in targeting Myc dependency in cancer cells. Thus,

our findings provide important insights into cancer and cancer stem cell biology and have

significant therapeutic implications.




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INTRODUCTION

Phosphatidylinositol 3’-kinase (PI3K)-AKT pathway is one of the most commonly

deregulated signaling pathways in human cancers (1). Genetic aberrations affecting this

pathway, such as activating mutations of PIK3CA or inactivation of PTEN, have been

identified in virtually all epithelial tumors (2). The 3-phosphoinositide-dependent protein

kinase-1 (PDK1) is known to be activated as a result of the accumulation of the PI3K

product phosphatidylinositol-3,4,5-trisphosphate (PIP3) and thus considered as an

important component of the PI3K pathway. PDK1 is a master regulator of AGC kinase

members, including AKT, p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-

induced protein kinase (SGK) and protein kinase C (PKC) family members, thus having

multiple roles in various physiological processes such as metabolism, growth,

proliferation and survival (3). In human cancers, PDK1 is thought to be constitutively

activated upon elevation of PIP3 owing to the loss of PTEN or gain of PIK3CA activity.

In addition, PDK1 deregulation in human malignancy can also be caused by gene

amplification or abnormal phosphorylation in cytosol and nucleus, such as colon cancer

and invasive breast cancer (4, 5).

One of the most defined PDK1 targets relevant in human cancer is AKT.

Specifically, PDK1 directly phosphorylates AKT on T308, but requires mTORC2-

induced AKT phosphorylation on S473 to confer a full activation (6). Given its

connection to AKT, PDK1 has been pursued as a critical anti-cancer target (7). However,

in view of the diversity of PDK1 substrates, additional downstream targets of PDK1 may

confer aberrant signaling heterogeneity and complexity in human malignance. Indeed, it

has been recently shown that inhibition of PDK1 has no significant effect on AKT




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signaling in a PTEN-deficient transgenic tumor mouse model (8) or breast tumor growth

(9), and oncogenic functions of PDK1 through substrates other than AKT, such as SGK3

(10), MAPK (11), or PKC� (12), have also been reported. In addition, our recent work

has shown that PDK1 is required for Myc protein accumulation in colon cancer cells

treated with mTOR inhibitor rapamycin (5), indicating a potential functional link of

PDK1 with Myc in oncogeneiss.

Myc is implicated in both cancer and stem cell self-renewal. The relationship

between stem cell and human cancers has become an important issue in cancer research

given that self-renewal is a hallmark of both cell types (13). Genes associated with

embryonic stem cell (ESC) identity, including pluripotency transcription factors,

Polycomb targets and Myc targets, have been observed in aggressive human cancers and

are associated with poor disease outcome (14). Moreover, the Myc associated molecular

network is strikingly similar between ESC and human cancer transcription programs (15),

and ectopic overexpression of Myc in differentiated somatic cells can induce both ESC

gene signature and properties of cancer stem cells (CSC) (16). These findings suggest

that activation of an ESC-like gene expression program in adult cells may confer self-

renewal to cancer cells or cancer stem cells. Notably, although the cancer associated

ESC–like gene regulation by transcription factors has been well documented, its

regulation by a druggable kinase-driven signaling pathway has yet to be identified.

In the present studies, we investigated PDK1-evoked key signaling events required

for oncogenic transformation. We identified that PDK1-PLK1-Myc pathway is a major

driver pathway conferring PDK-induced transformation and its existence is readily

evident in human cancers. We further show that PDK1-PLK1-Mys signaling drives an




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ESC-like gene expression signature relevant in human cancers and is robust in inducing

CSC phenotype. It also involves in resistance to mTOR inhibitor in colorectal cancer

cells. These findings provide important insights into the cancer and cancer stem cell

biology and potential new treatment for targeting Myc-dependency in human cancers

RESULTS

PDK1-Induced Myc Protein Induction Confers Oncogenic Transformation

As the first step to investigate the differential signaling pathways activated by PDK1 or

PI3K in tumorigenesis, we compared the transforming capacity of PDK1 and PI3K by

using the in vitro transformation assay that measures the anchorage-independent growth

in soft agar. We began with semi-transformed human embryonic kidney epithelial cells

(HEK) that express a low level of activated HRasV12 (HEK-TERV)(17) and infected

them with retroviral vectors expressing PDK1, Myc, a constitutively activating mutant of

PIK3CA (E545K) or PTEN small hairpin RNA (shRNA), resulting in stable cell lines

designated as HEK-PDK1, HEK-Myc, HEK-E545K or HEK-shPTEN cells, respectively.

The transformation assay results showed that they were all able to induce cellular

transformation, although PDK1- or Myc-induced colonies appeared to be larger in size as

compared with that of E545K- or shPTEN-expressing cells (Fig. 1A and Supplementary

Fig. S1A). Consistent with our previous report showing a post-translational Myc

induction by PDK1 (5), we detected a marked protein accumulation of Myc in HEK-

PDK1 cells but not in HEK-E545K or HEK-shPTEN cells (Fig. 1B) which was not due to

the induction of Myc mRNA level (Supplementary Fig. S1B). We also show that the

kinase activity of PDK1 is required for transformation as well as Myc protein induction




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as a kinase-dead mutant of PDK1 (PDK1 K100N) (9) induced neither the transformation

nor the Myc accumulation (Supplementary Fig. S1C).

A survey of known AGC substrates of PDK1 revealed that PDK1 also induced a

strong phosphorylation of PKC� and a modest increase of phosphorylated AKT (T308).

The other known PDK1 substrates, including SGK1/3 and S6K, were not activated, nor

AKT phosphorylated at S473, which is required for a full activation of AKT. In contrast,

E545K overexpression induced strong phosphorylations of AKT (at both T308 and S473)

as well as the downstream substrates FOXO1 and FOXO3A (Fig. 1B). Thus, the

remarkable observation that PDK1 induces transformation in the presence of a weak

AKT activation suggests a potential more functional role of Myc in this process. Indeed,

RNA interference-mediated knockdown of Myc resulted in much reduced transformation

of HEK-PDK1 cells, but not in HEK-E545K cells (Fig. 1C), demonstrating a Myc-

dependency for PDK1-induced transformation. Moreover, in a series of dose-response

analysis (see Supplementary Fig. S1D&E), HEK-PDK1 cells, compared with HEK-

E545K cells, were much more sensitive to small molecule PDK1 inhibitors BX795 and

BX912 (Fig. 1D, Left and Supplementary Fig. S1D). In contrast, E545K-transformed

cells were much more sensitive to the PI3K inhibitor GDC-0941 and the AKT inhibitor

MK2206 and GSK690693 (Fig. 1D, Left and Supplementary Fig. S1E). Consistent with

these effects, BX795 reduced Myc accumulation but had only a modest effect on AKT.

By contrast, GDC-0941 or MK2206 easily abolished phosphorylations of AKT in HEK-

E545K cells, but had no such effects on Myc inhibition (Fig. 1D, Right). These results

demonstrated the differential pathway dependency for the two transformed cell systems.

Interestingly, Myc-transformed cells were also sensitive to BX795 (Fig. 1D, Left), which




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is consistent with the observation that BX795 was able to eliminate the exogenous Myc

in these cells (Fig. 1D, Right). Altogether, these data show that PDK1-induced

transformation depends more on Myc, but less on AKT signaling, when compared with

E545K-driven transformation. The data also suggest that Myc-dependent cells become

sensitive to the PDK1 inhibitor, regardless of PDK1 status, which reveals a PDK1-

dependency in Myc-driven cells. PDK1-induced Myc activation upon transformation was

also observed in immortalized human mammary epithelial cells (HMEC) and prostate

epithelial cells (RWPE-1) (Fig. 1E and F), suggesting that the Myc activation by PDK1 is

not restricted to HEK but occurs in multiple epithelial lineages.

To demonstrate the physiological relevance of PDK1-Myc connection in human

cancers, we show that PDK1 knockdown was able to eliminate Myc expression in a

variety of human cancer cell lines (Fig. 1G). Moreover, in a panel of breast cancer cell

lines in which the Myc-dependent viability has been previously characterized (18),

BX795 treatment resulted in similar Myc depletion in all these cells (Fig. 1H) but

preferentially reduced the cell viability of Myc-dependent breast cancer cell lines (MDA-

MB-231, Hs578T, and SUM159PT) as compared to Myc-independent breast cancer cell

lines (T47D and BT474)(Fig. 1I). Of notice, in these cell lines BX795 seemed to inhibit

AKT and FOXO3A phosphorylations in a cell-dependent manner (Fig. 1H). Taken

together, these results show a potential role of PDK1 activity towards Myc regulation

which is therapeutically implicated for Myc-driven tumors.

Synthetic Lethal Screening Identifies PLK1 as a Crucial Downstream Effector of

PDK1 for Myc Induction and Cancer Cell Survival




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To investigate whether or not there are downstream kinase(s) of PDK1 that is crucial for

Myc induction and cell transformation, we performed a screen for kinases that, when

pharmacologically inhibited, selectively kill PDK1-transformed cells. Among 60 small

molecule protein kinase inhibitors we have screened, we found that two PLK1 inhibitors

(BI2536 and GW843682X), one MEK inhibitor (PD0325901), and one ALK inhibitor

(NVP-TAE684), PD180970, one Brc-Abl inhibitor (PD180970) and one tyrosine kinase

inhibitor (Sunitinib) showed a preferential inhibitory effect on the viability of HEK-

PDK1 cell as compared to the control cells (Supplementary Fig. S2A). The two PLK1

inhibitors were further validated in a secondary screen and were thus chosen for further

study (data not shown). Further analyses in all the three epithelial systems showed that

PDK1-transformed cells were much more sensitive to the PLK1 inhibitors compared to

vector control or E545K-transformed cells (Fig. 2A and B). This finding reveals a

possible role of PLK1 in PDK1-induced transformation. Indeed, western blot analysis

showed an induction of PLK1 phosphorylation in all the three PDK1-transformed cell

lines, but not in E545K- or Myc-transformed cells (Fig. 2C). Similar to PDK1 inhibitor

BX795, BI2536 treatment resulted in strong colony growth inhibition in both PDK1-and

Myc-transformed cells, but not in E545K-transformed cells (Fig. 2D and Supplementary

Fig. S2B). Furthermore, like BX795, PLK1 inhibitor BI2536 or GW843682X was able to

eliminate endogenus Myc in HEK-PDK1 cells but also the exogenous Myc in HEK-Myc

cells (Fig. 2E). This finding suggests that the exogenous Myc is also sensitive to the

perturbation of the basal level of PDK1-PLK1 signaling. Accordingly, in both HEK-

PDK1 and HEK-Myc cells, but not in HEK-E545K cells, BI2536 treatment resulted in

strong apoptosis, as demonstrated by both FACS analysis (Fig. 2F) and increased caspase




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3 activity (Supplementary Fig. S2C), while E545K-transformed cells mainly displayed

G2/M arrest, a typical feature related to a mitotic effect following PLK1 inhibition

(Supplementary Fig. S2D). Furthermore, to confirm the PLK1-specific effect of BI2536,

PLK1 depletion by three independent siRNAs gave rise to similar effects on endogenous

and exogenous Myc and apoptosis in PDK1 or Myc-driven cells (Fig. 2G). These

findings suggest a crucial role for PDK1-PLK1 signaling in regulating Myc and cancer

cell survival. Consistent with the in vitro data, xenograft tumors derived from HEK-

PDK1 cells were highly sensitive to BI2536 treatment and displayed a strong tumor

regression following just two dosages, while the same treatment only induced tumor

growth inhibition in E545K-derived xenograft tumors (Fig. 2H).

We further show that the PLK1 regulation of Myc is not limited to transformed

cells but physiologically relevant in human cancers as either PLK1 knockdown or BI2536

treatment resulted in endogenous Myc protein depletion in various cancer cell lines

without changing Myc mRNA level (Supplementary Fig. S3A-C). In addition, a time

course analysis indicates that BI2536 treatment can result in Myc depletion as early as 8

hrs, concomitant with an early G2/M arrest (Supplementary Fig S3D), indicating that

Myc downregulation is unlikely to be a result of the secondary effect of cell cycle

change. BI2536 treatment also resulted in more effective growth inhibition in Myc-

dependent breast cancer cell lines compared to Myc-independent cells (except MDA-

MB-231) (Fig. 2I). These results further support a role of PDK1-PLK1 signaling in

supporting Myc-driven tumorigenesis.

PDK1 Induces PLK1 Phosphorylation in Human Cancer Cells




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We next sought to determine whether or not the PLK1 activation by PDK1 seen in

transformed cells represents a finding that is physiologically relevant in human cancer

cells. To achieve this, we first used the colon cancer HCT116 and DLD1 cells in which

PDK1 is genetically knocked out (19). To facilitate the detection of phosphorylation of

PLK1, which is a mitotic kinase, cells were first synchronized by double-thymidine block

and then released into the cell cycle progressively (Fig. 3A and Supplementary Fig. S4A).

In PDK1 wild-type cells, we noticed progressive induction of PDK1 phosphorylation

upon cell cycle progression into mitosis as indicated by elevated levels of phosphor-

histone H3, which was accompanied by a similar pattern of PLK1 phosphorylation,

whereas in PDK1-/- counterparts we detected a much more deficient PLK1

phosphorylation and Myc accumulation but not the phosphorylation of the PLK1-related

kinase Aurora A (20, 21)(Fig. 3A and Supplementary Fig. S4A).

We also probed the changes of AKT-mTOR pathway in these cellular contexts.

Of notice, compared with p-PLK1, p-AKT (T308) was only modestly changed in this

condition in PDK1-/- cells, while p-AKT( S473) and p-FOXO3A were even enhanced in

both PDK1-/- cell lines (Fig. 3A). This could be due to the inhibition of S6K in PDK1-/-

cells, leading to a feedback activation of p-AKT (S473). In contrast, in a different

condition where cells were serum starved and then stimulated with growth factors for

early time points, we saw a clear p-AKT-(T308) inhibition in HCT116 PDK-/- cells

(Supplementary Fig. S4B). Thus, PDK1 regulates p-PLK1 and p-AKT (T308) in different

growth conditions. To further consolidate the data, we also performed PDK1 knockdown

by shRNA in MDA-MB-231 cells. The result shows again that the PDK1 knockdown

resulted in ablation of PLK1 phosphorylation and Myc accumulation (Fig. 3B), as well as




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deficient entry into mitosis (Fig. 3C). These data consolidated a role of PDK1 in driving

PLK1 and Myc activation not just in a confined system but also in cancer cells.

Furthermore, in multiple cancer cell lines treated with BX795, GDC0941 or

MK2206 upon double-thymidine block and release (Supplementary Fig. S4C), we saw

that BX795 always blocked PLK1 phosphorylation and Myc accumulation, but inhibited

AKT phosphorylation in a cell line dependent manner (for example, AKT

phosphorylation is not affected by BX795 in MDA-MB-231 cells). By contrast,

GDC0941 and MK2206 consistently inhibited AKT phosphorylation in each of these cell

lines, but had little effect on PLK1 and Myc. Together, these data demonstrate the

physiological relevance of AKT-independent PDK1-PLK1-Myc signaling in cancer cells.

We next investigated whether or not PLK1 is a potential substrate of PDK1.

PDK1 is known to regulate AGC kinases. Protein domain analysis indicates that the

kinase domain of PLK1 is part of the AGC kinase family (Fig. 3D). Interestingly, the

amino acid sequence surrounding the Thr210 contains a consensus motif for PDK1 which

is found in many known PDK1 substrates (Fig. 3D), thus enhancing the possibility that

PLK1 could be a potential substrate of PDK1. Indeed, co-transfection of PDK1 and

PLK1 into 293T cells, followed by PLK1 immunoprecipitation, showed that PDK1

enhanced the phosphorylation of both the endogenous and exogenous PLK1, which was

abolished when cells were treated with BX795, BX912, but not Aurora A inhibitor

VX680 (Fig. 3E). This suggests that PDK1-induced PLK1 phosphorylation was unlikely

to be an indirect effect of Aurora A, which might be co-immunoprecipiated with PLK1.

Furthermore, in an in vitro kinase assay using endogenous PLK1 immunoprecipitated

from DLD1 as a substrate, recombinant PDK1 added in the kinase assay induced the




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PLK1 phosphorylation at T210, which was markedly reduced in cells treated with BX795

(Fig. 3F), indicating that the recombinant PDK1 can directly induce endogenous PLK1

phosphorylation in vitro. Importantly, in cells treated with VX680, where the PLK1

phosphorylation was greatly reduced as expected, recombinant PDK1 still boosted the

PLK1 phosphorylation in the in vitro kinase assay (Fig. 3F). This further excludes the

possibility that PDK1 may induce PLK1 phosphorylation indirectly through Aurora A

kinase. Finally, in an in vitro kinase assays using both PDK1 and PLK1 as recombinant

proteins, we show that PDK1 induced a strong PLK1 phosphorylation, which was

blocked by BX795, BX912, but not by VX680 (Fig. 3G). Collectively, these experiments

provided evidence that PDK1 directly regulates PLK1 in human cancer cells.

PLK1 Directly Interacts with Myc and Induces Myc Phosphorylation in a PDK1-

Dependent Manner

We next investigated whether PLK1 directly regulates Myc. Through co-

immunoprecipitation (co-IP) assay, we demonstrated an interaction between both

exogenous PLK1 and Myc in 293T cells (Fig. 4A). We also showed that the endogenous

interaction between the two proteins occurs in HEK-PDK1 cells as well as in various

cancer cell lines (Fig. 4B and C). We further showed that PLK1 kinase activity is

required for the Myc protein accumulation as the wild type PLK1, but not the kinase dead

mutant, induced strong Myc accumulation (Fig. 4D). Crucially, in vitro kinase assays

using either recombinant PLK1 (Fig. 4E) or endogenous PLK1 pulled down from the

cancer cells (Fig. 4F) demonstrated a robust induction of S62 phosphorylation of

recombinant Myc but not T58 phosphorylation, which was reduced in the presence of




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BI2536. Importantly, the endogenous PLK1 kinase activity towards Myc phosphorylation

was strongly abolished in cells treated with BX795 (Fig. 4G) or in PDK1-/- cells (Fig.

4H). Thus, these results not only showed a direct phosphorylation of Myc by PLK1, but

also showed that PLK1 activity on Myc is crucially dependent on PDK1. Together, these

data reiterate the operation of PDK1-PLK1-Myc signaling in cancer cells.

PDK1-PLK1-Myc Signaling Drives Cancer Initiating Cell Maintenance and Self-

Renewal

During culturing of these transformed cells, we noticed that HEK-PDK1 cells, and to a

lesser extent, HEK-Myc cells, displayed distinct morphologies from HEK-E545K cells

and once they became confluent in culture, started to form semi-attached 3D clusters on

the plate (Fig. 5A, Upper), suggesting that they displayed tumorigenic and stem cell-like

properties. This feature, however, was not observed in E545K-transformed cells (Fig.

5A). Given a known role of Myc in inducing ESC- or CSC-like phenotypes in

differentiated somatic cells (16), it raises a possibility that PDK1, which activates Myc,

may have a similar capacity in inducing CSC-like behavior. This hypothesis was first

tested by using an in vitro assay for spheroids formation in serum-free suspension culture,

a property associated with cancer stem/progenitor cells (22). We observed that PDK1-or

Myc transformed HEK or HMEC cells formed large and abundant non-adherent

tumorspheres after 7 days of growth in suspension culture, whereas E545K-transformed

cells were only able to generate low number of small spheres (Fig. 5A, Bottom and

Supplementary Fig. S5A). These spheres were able to reform a monolayer when placed

back to a tissue culture plate containing serum-rich medium (Fig. 5B). Furthermore, after




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dispersion into single cells, PDK1 or Myc-transformed HEK or HMEC cells reformed

spheres with increasing enrichments for at least 4 passages (Fig. 5C and Supplementary

Fig. S5B), indicating a gain of self-renewal capacity that resembles a stem cell-like

property.

To demonstrate the tumor-initiating capacity of these transformed cells in vivo,

we next injected these cells at different numbers into the flank of BALB/c nude mice.

Strikingly, 1000 HEK-PDK1 cells were sufficient to generate tumors in all 6 mice as

early as 2 weeks (Fig. 5D, left). HEK-Myc cells seemed to be less tumorigenic and

required 10,000 cells to generate a similar size of tumors. By contrast, 10,000 HEK-

E545K cells were unable to induce tumors in the mice (Fig. 5D). A further experiment

shows that as low as 100 PDK1 cells were sufficient to give rise to xenograft tumors,

whereas 3 x 106 E545K cells were required to generate observable tumors by 28 days

(Fig. 5D, right). Importantly, PDK1-associated primary xenograft tumors were self-

renewable, as determined by the ability to form secondary and tertiary tumors using as

low as 100 xenograft tumor cells (Fig. 5E). These in vitro and in vivo data demonstrated a

strong tumorigenicity of PDK1-transformed cells with self-renewal capacity.

We also tested the ability of PDK1 in inducing mouse embryonic fibroblasts

(MEFs) reprogramming. To achieve this, we used p53–deficient MEFs, as

immortalization by p53 inactivation has been shown to enhance MEF reprogramming

efficiency (23, 24). Again, PDK1 but not E545K was also able to induce Myc activation,

as well as tumorsphere formation in immortalized p53-/- MEFs (Figure S5C and S5D). In

the PDK1-sphere populations we detected strongly increased expressions of ES

pluripotency factors Sox2 and Oct4 as assessed by both qPCR and confocal imaging




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compared to the monolayer growth (Figure S5E and S5F). When these MEF-PDK1cells

were cultured in ESC medium containing the differentiation inhibitor LIF (leukemia

inhibitory factor), MEF-PDK1 cells formed colonies resembling the ESC-like

morphology and were alkaline-phosphatase (AP) positive (Figure S5G), though we found

that these colonies were unable to maintain the ES-like morphology in the subsequent

passages, probably due to an incomplete reprogramming. Thus, in both human epithelial

cells and MEFs, PDK1 is able to induce PLK1 and Myc activation and ESC-like

property.

Aberrant high PDK1 activity has been shown in invasive and metastatic breast

tumor samples (25). To demonstrate the capacity of the PDK1-PLK1-Myc pathway in

regulating cancer stem cells, we utilized the highly invasive breast cancer MDA-MB-231

and SUM159PT cells that contain a high percentage of CD44+/CD24-low CSC-like

population (26). Intriguingly, PDK1-PLK1-Myc signaling was found to be enriched in

CD44+/CD24-low cells compared to the non-CD44+/CD24-low cells (Fig. 5F).

Knockdown of PDK1/PLK1 or treatment with their corresponding inhibitors

BX795/BI2536, resulted in marked reduction of CD44+/CD24-low populations (Fig. 5G,

H and I). By contrast, PI3K/AKT inhibitors GDC-0941 and MK-2206 were unable to do

so (Fig. 5I). Corresponding to the reduced CD44+/CD24-low cells, PDK1 or PLK1

inhibition either by gene knockdown or inhibitor treatment resulted in marked inhibition

of tumorsphere formation in MDA-MB-231 cells (Fig. 5J).

PDK1 Activates ES or CSC-like Transcriptional Programs




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It is known that Myc is able to activate ESC-like transcriptional programs in adult

epithelial cells, resulting in a CSC-like phenotype in the appropriate genetic context (16).

To characterize the transcriptional program underlying the PDK1-induced CSC-like

behavior, we compared the gene expression profiles in HEK-PDK1, -Myc or -E545K

cells. Significant Analysis of Microarray (SAM) identified 1750, 1080 and 297

differentially expressed genes in these transformed cells when compared to non-

transformed control cells, respectively (FDR < 0.05, p < 0.01; Supplementary Table S1-

3). Gene Venn Diagram analysis shows that HEK-PDK1 shared a robust transcriptional

program with HEK-Myc, but had little overlap with HEK-E545K (Fig. 6A). In addition

to the PDK1 and Myc common gene set, PDK1 also regulates a unique set of 784 genes.

We further stratified the PDK1- or Myc-regulated genes into 889 upregulated and 1151

downregulated genes via gene cluster analysis (Fig. 6B). Notably, a number of well-

known genes implicated in ESC pluripotency or maintenance, including SOX2, LIN28B,

SALL4 and EZH2 (27), or CSC, including EPCAM, ALDH1A and S100A4 (28), were

upregulated in both HEK-PDK1 and HEK-Myc cells, but not in HEK-E454K cells (Fig.

6B). JAG2, which was recently shown to be a Myc target (29) with a role in modulating

CSCs, was also markedly induced by PDK1 and Myc but not by E545K. In addition, a set

of secreted inhibitors of autocrine signaling, including DKK1, SFRP1, and BMP4, whose

reduction has been recently shown by Weinberg’s group to enable self-renewal of

epithelial cells (30), were strongly repressed in PDK1 and Myc cells but not in E545K

cells. CD24, a negative selection marker for CSCs (31), was also selectively repressed in

PDK1 and Myc cells. The array results of selected genes were further validated by qRT-

PCR (Fig. 6C) and Western blotting (Fig. 6D). Notably, many genes co-regulated by




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PDK1 and Myc, including SOX2, EPCAM, JAG2 and S100A4, were more affected by

PDK1 than Myc. In total, we identified 668 genes showing such a pattern (Fig. 6E and

Supplementary Table S4), which is consistent with a more robust role of PDK1 than Myc

in tumorigenesis.

We also investigated the changes of microRNAs that are Myc-associated and

implicated in ESC self-renewal. LIN28B is a known Myc target and is able to inhibit the

biogenesis of the let-7 family microRNAs (32). Inhibition of let-7 miRNAs has been

shown to enhance reprogramming of somatic cells to induced pluripotent (iPS) cells (33).

Myc also transactivates the mir-17-92 cluster, which is also implicated in ESC

maintenance (34). Consistent with Myc and LIN28B elevation, we detected a marked

upregulation of miR-17-92 and down-regulation of let-7s in PDK1 and Myc cells, but not

in E545K cells (Fig. 6F). Since let-7 suppresses its own negative regulator LIN28B (33),

it is likely that PDK1 enforces a feedback loop via Myc-LIN28B-mediated Let-7

downregulation to support the self-renewal program. Lastly, we show that BI2536

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 in the PDK1-mediated CSC gene signature. These findings indicate that PDK1 is

able to evoke multiple transcriptional programs that coordinately induced a remarkable

reprogramming towards a state resembling CSCs. In this process, Myc is one important

factor but not the only one that modulates the reprogramming.

PDK1-Induced CSC-like Gene Signature is Relevant to Human Cancers and is

Associated with Aggressive Tumor Behavior




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Aberrant gene expression associated with ES cell identity, including ESC genes, Myc

targets and Polycomb targets, have been found in poorly differentiated tumors (14, 16,

35). By further interrogating several previously published datasets collectively, we show

that the above ESC-related genes were significantly enriched in the PDK1-dependent

transcriptome, including upregulation of 97 ESC-expressed genes and downregulaton of

182 Polyccomb targets (PRC genes) (Supplementary Fig. S6A and Table S5). In contrast,

the E545K-associated transcriptional program displayed a distinct gene set that is not

significantly associated with ESCs (Supplementary Fig. S6A).

We next determined whether PDK1-driven ESC-like gene expression is of clinical

relevance to human malignance. Gene set enrichment analysis (GSEA)(36) of several

previously published data sets showed that PDK1-induced ESC-like genes were found to

be significantly enriched in colon and lung tumor samples as compared to the normal

controls, while the Polycomb targets were reversely correlated in these samples

(Supplementary Fig. S6B). Moreover, in breast cancer, deregulation of these genes was

significantly correlated with the high-grade tumors as compared to the low-grade tumors

(Supplementary Fig. S6C). This indicates that aberrant expression of these PDK1-

regulated ESC genes is associated with malignant progression from normal to aggressive

tumors. In addition, we also demonstrated that the PDK1-regulated ESC-like gene

signature was associated with poor disease outcome as shown in the survival analysis of

breast and lung cancer cohorts (Supplementary Fig. S6D), providing a prognostic value

of these genes. Together, these findings demonstrate that the PDK1-activated ESC-like

gene signature we identified from the in vitro culture system is clinically relevant to




19

human cancers arising in distinct tissues and support a link of PDK1-Myc signaling to

aggressive cancer behavior.

BI2536 Synergizes with PI3K-mTOR Inhibitor BEZ235 to Induce Robust Apoptosis

and Tumor Growth Inhibition in CRC

We have previously shown that mTOR inhibition by rapamycin or mTOR/Raptor

knockdown induces Myc accumulation in CRC, which can be inhibited by PDK1

inhibition, resulting in rapamycin sensitization (5). As our data now indicate that PLK1 is

required for PDK1-Myc signaling, together with our further observation that PLK1 is

highly expressed in CRC tumors compared to adjunct normal regions (Supplementary

Fig. S7A, B), we hypothesized that the PLK1 inhibitor could also sensitize CRC cells to

mTOR inhibitors through abolishing mTOR inhibitor-induced Myc activation. Classical

mTOR inhibitors like rapalogs are known to induce compensatory feedback activation of

PI3K-AKT due to S6K inhibition. BEZ235, a dual PI3K-mTOR kinase inhibitor is able

to overcome the feedback AKT activation and is currently being tested in several clinical

trials either as a single agent or in combination with other therapeutics. Unlike

Rapamycin treatment which induced both AKT and Myc activation in CRC cells,

BEZ235 did not induce AKT activation but retained the ability to induce Myc (Fig. 7A).

Of notice, neither drugs induced ERK activation in CRC, which is however often seen in

breast cancer cells (37). As expected, BI2536 co-treatment effectively removed BEZ235-

induced Myc induction (Fig. 7B). In these cells, BI2536 or BEZ235 alone failed to

induce significant apoptosis, but their combination, which resulted in inhibition of both

Myc and p-4E-BP1, induced a massive apoptosis, as evidenced by strong detection of




20

PARP cleavage (Fig. 7B), cells in Sub-G1 (Fig. 7C), and caspase 3 activation

(Supplementary Fig. S7C). The combinatorial effect was synergistic as shown by

combination index analysis (Supplementary Fig. S7D) and further confirmed by time-

course analysis of cell viability (Fig. 7D) and long term colony formation assay

(Supplementary Fig. S7E). Finally, to assess the potential of the combination strategy in

vivo, SW480 and HT15 cells were injected subcutaneously into nude mice to establish

tumor xenografts. We demonstrated that BEZ235 also induced Myc accumulation in the

xenograft tumors which can be inhibited via combination with BI2536 (Supplementary

Fig. S7F). Accordingly, the combination treatment induced synergistic tumor growth

inhibition compared to the single agent treatment, validating the in vitro findings (Fig.

7E).

Like BEZ235, a specific mTOR inhibitor PP242 (38) also generated similar

results on Myc, p-4EBP1 and apoptosis when combined with BI2536 (Supplementary

Fig. S8A, B). On the contrary, BI2536, though it also blocked Rapamycin-induced Myc

accumulation (Supplementary Fig. S8C), did not enhance apoptosis (Supplementary Fig.

S8D), only potentiated the anti-proliferation effect and xenograft tumor growth inhibition

(Supplementary Fig. S8E, F). This is probably due to the inability of rapamycin to block

4EBP1 phosphorylation (Supplementary Fig. S8C) as previously shown. 4EBP1, but not

S6K, has been recently shown to be the key effector of the mTOR pathway responsible

for cell proliferation and survival (39) and additional inhibition of 4EBP1

phosphorylation seems to be required for apoptosis induction in response to the AKT

inhibitor (40). Thus, the simultaneous inhibition of both Myc and 4EBP1 phosphorylation

upon combination of BI2536 and BEZ235 seemed to be crucial for apoptosis induction of




21

CRC cells. Taken all the data together, we conclude that the combination of BI2536 and

PI3K-mTOR dual inhibitors like BEZ235 may represent a promising treatment strategy

for CRC.

DISCUSSION

PDK1 Regulation of PLK1-Myc Signaling in Human Cancer Cells

Although PI3K-AKT signaling has been considered to be the main signaling pathway

associated with PDK1 in oncogenesis, our study uncovers another arm of signaling that

routs to PLK1-Myc to confer malignant phenotypes. Importantly, the pathway we

identified using a chemical genetic approach with a PDK1-transformed cell line has been

validated to be relevant in human cancers as demonstrated in multiple cancer cell lines

derived from various tissue types. Although in our system we detected AKT

phosphorylation at T308 by PDK1, we did not see AKT phosphorylation at S473 which

is required for a full AKT activation (6). This is in contrast to PI3K-transformed cells

where both AKT phosphorylations are strongly induced. This observation is consistent

with a recent report showing that PDK1-deficiency in colon cancer cells has only a

modest effect on p-AKT (T308) and had no effect on p-AKT (S473) (19). Our data thus

indicate that PDK1 signaling might be wired differentially in certain oncogenic contexts

to confer growth advantage that becomes less dependent on AKT. Indeed, previous

reports have shown that PDK1 is unlinked to PI3K signaling in a PTEN-deficient tumor

model (8) and can route through the AKT-independent pathway for cell survival in some

cancer cell lines (10). As PDK1 has attracted much attention as a potential therapeutic

target in cancer, we propose that Myc can be an alternative pharmacodynamic marker for




22

the evaluation of small molecule PDK1 inhibitors under preclinical and clinical

development.

Therapeutic Targeting of PDK1-PLK1 Signaling in Myc-dependent Tumors

An intriguing finding of this study is the identification of the crucial role of PDK1-PLK1-

Myc signaling for cancer cell survival. We provide evidence that PDK1 induces PLK1

phosphorylation and PLK1 binds to and induces Myc phosphorylation and protein

accumulation widely in cancer cells. A previous report has shown that PLK1-induced

Myc phosphorylation is required for SCF�TrCP-mediated Myc protein stabilization during

late stage cell cycle progression (41). We further show here that PLK1 can directly bind

to Myc. Regardless of whether or not PDK1-PLK1 signaling regulates Myc stability

through a similar or distinct mechanism, the direct regulation of Myc by PDK1-PLK1

signaling immediately suggested a therapeutic approach targeting Myc-driven tumors.

Indeed, our data show a preferentially killing of small molecule inhibitor of PDK1 or

PLK1 in Myc-dependent breast cancer cells compared with Myc-independent breast

cancer cells. Given that a clinical inhibitor of Myc is not available, small molecule

inhibitors like BI2536, which is currently in late stage of clinical trial, may provide an

alternative anti-Myc strategy. Given PLK1 is often found to be overexpressed in human

cancers (42), therapeutic targeting of PLK1 may yield a more favorable therapeutic index

in Myc-associated tumors.

Role of PDK1-PLK1-Myc Signaling in Driving Tumor Initiating Cells




23

The main characteristic of the PDK1-induced transformation is that it is able to induce

both genotype and phenotype of CSCs that has been proposed to account for tumor

initiation, progression and chemo-resistance (13, 31). We show that as low as 500 PDK1-

transformed cells can induce robust tumorigenicity and the PDK1 activates clinically-

relevant transcriptional programs associated with poor disease outcome. In addition,

PDK1 or PLK1 inhibition also resulted in disruption of both embryonic and adult stem

cell self-renewal while inducing differentiation. Activation of an ESC-like signature and

an ESC-like phenotype in differentiated somatic cells also indicates that the ES program

can be reactivated during the course of tumor progression and is not necessarily inherited

from a stem cell-of-origin. This notion is consistent with recent report from Weinberg’s

group showing spontaneous CSC-generation from mammary epithelial cells (43).

Furthermore, consistent with a previous report that PDK1 is hyperactivated in

invasive and metastatic breast cancer (25), we show that PDK1 or PLK1 inhibition in

highly invasive breast cancer MDA-MB-231 cells resulted in depletion of CSC-like

CD44+/CD24-low populations and accordingly strongly reduced tumorsphere formation,

while PI3K-AKT inhibition did not have such effects. Thus, small molecule inhibition of

PDK1-PLK1-Myc signaling for elimination of CSCs may provide a targeted therapy to

overcome recurrence of aggressive breast tumors following chemotherapy.

Combination of PLK Inhibitor and PI3K-mTOR Inhibitor for CRC

An additional therapeutic application of our studies is the identification of strategies to

overcome resistance to mTOR-targeted therapy in CRC. Drug resistance and tumor

recurrence is the main cause of patient relapse, possibly owing to recurrence of cancer




24

stem cells. We have previously discovered that mTOR inhibition induces Myc activation,

a compensatory effect mitigating the anti-proliferative effect of mTOR inhibitors in CRC

(5). We now show that PLK1 inhibitor blocks mTOR inhibitor-induced Myc activation,

providing a rational approach to develop a new combination therapy for CRC.

Specifically, low dose of PLK1 inhibitor BI2536 plus PI3K-mTOR dual inhibitor

BEZ235 induced massive apoptosis in CRC cells and a synergistic loss of colony

formation, suggesting that this strategy might be useful in CRC. Given both drugs are in

late stage clinical trials, we hope that our findings could spur clinical trials in CRC for

combination of BEZ235 and BI2536 to improve the therapeutic outcome.

MATERIALS AND METHODS

Constructs and Reagents

Human full-length PDK1, Myc, PIK3CA-E545K and PLK1 were cloned into PMN-

IRES-GFP retroviral vector and introduced into human epithelial cells and MEFs. All

kinase inhibitors used in this study were obtained from Axon Medchem. Information for

plasmid DNA vectors and stable cell line construction are provided in Supplemental

Materials and Methods

Cell Cultures

Cell cultures and various cellular assays are described in Supplementary Materials and

Methods. All cancer cell lines were purchased from American Type Culture Collection

(ATCC) (Manassas, VA) and No authentication of cell lines was done by the authors.

Mouse Experiments




25

All of the experiments in xenografts are described in Supplemental Materials and

Methods.

Immunobloting, immunoprecipitation and in vitro kinase assays Details are described in Supplemental Materials and Methods

Gene Expression, Data Analysis and Real-Time PCR Analysis

The microarray hybridization was performed using the Illumina Gene Expression Sentrix

BeadChip HumanHT-12_V4 (Illumina) and the data was analyzed using the GeneSpring

GX 11.0.2 (Agilent Technologies). Detailed information can be found in Supplemental

Materials and Methods. Primers used in real-time PCR analysis are described in Table

S6.

Statistical Analysis

PDK1 regulated ESC-like and PRC gene signature definition was described in

Supplementary Experimental Procedures. Gene Set Enrichment Analysis (GSEA) (36)

was conducted to assess the degree of correlation between PDK1-regulated gene

signatures and cancer phenotypes on different human patient. Survival curves were

calculated using the Kaplan-Meier survival analyses and the quantiles-rank test. Detailed

statistical analysis is included in Supplemental Information. Data are presented as mean ±

SEM, unless otherwise stated. A student’s t test was used to compare two groups for

statistical significance analysis.

Accession number




26

The microarray data are deposited into the Gene Expression Omnibus (GEO) with the

accession number GSE30669.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Materials and Methods, 8 figures, and

six tables.

ACKNOWLEDGEMENTS

We thank Dr. William C. Hahn for the HEK-TERV cells. We thank Dr. Fu Zheng for the human

PLK1 plasmids and Dr. Luca Primo for the PDK1 kinase-dead mutant construct. This work was

supported by the Agency for Science, Technology and Research of Singapore.

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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 non-

targeting siRNA (siNC) or Myc siRNA, respectively. * P<0.01.

(D) Soft-agar growth of HEK-PDK1, HEK-E545K and HEK-Myc cells treated with

BX795 (2.5 μM), GDC-0941 (0.5 μM), or MK2206 (0.5 μM) for 14 days. Right panel

shows the changes of 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

�M) for 24h.

(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 Myc-independent

(T47D and BT474) to BX795 treatment.

All the data in the graph bars represent mean ± SEM, n=3.




30

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 cell 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 nM BI2536

for 4 days.

(C) Immunoblot analysis of PLK1 in indicated cell lines.

(D) Soft-agar growth of indicated cell lines treated with 10 nM BI2536 for 14 days.

(E) Immunoblot analysis in HEK-PDK1 and HEK-Myc cells treated with BI2536 and

GW843682X as indicated concentration for 24 hr.

(F) Apoptosis by sub-G1 analysis of indicated cell lines treated with 10 nM BI2536 for

48 hr.

(G) Apoptosis of indicated cell lines treated with NC or PLK1 siRNAs for 48 hr (Upper)

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 Experimental Procedures. Data are

means ± 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 Myc-independent

(T47D, BT474, MCF-10A and HMEC) to BI2536 treatment.

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.




31

(D) PLK1 protein domain analysis (upper) 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 �M BX795, 5.0 �M BX912 and

1.0 μM VX680 treatment for 24 hr.

(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

μM BX795 or 1.0 μM VX680 for 8 hr. PLK1 IP-kinase assay was performed 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 μM BX795, 1.0 μM BX912 and 1.0 μM VX680.

Figure 4 PLK1 interacts with Myc and induces Myc phosphorylation

(A) Co-immunoprecipitation analysis in 293T cells transfected with ectopic PLK1, Myc,

or both.

(B) Co-immunoprecipitation analysis of endogenous PLK1 and Myc in HEK-Vector and

HEK-PDK1 cells.

(C) Co-immunoprecipitation 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 WT or kinase dead mutant of PLK1 (KD) in the absence or presence

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 �M BX795.

(H) Immnoblot analysis of in vitro kinase assay using immunoprecipitated PLK1 from

DLD1 and DLD1 PDK1-/- cells.




32

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 in upper panel. Lower panel shows 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 DMEM, 10% FBS and the sphere reformed a monolayer for 48

hr. 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 4 passages.

(D) Xenograft tumor growth in nude mice. HEK-PDK1, Myc or E545K injected with

indicated cell numbers were shown. Data are means ± SEM.

(E) Xenograft tumor formation frequencies of tumor-initiating cells derived from the

first, 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 profiles 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 means ± SEM (n=3).

Figure 6 PDK1 evokes ESC-like gene expression profile

(A) Venn diagram showing the overlapping of differentially expressed genes in HEK-

PDK1, Myc or E545K as compared with HEK-vector control cells. .

(B) Heat map of differentially expressed genes in HEK-PDK1, Myc or E545K cells.




33

(C) qRT-PCR analysis of representative genes in HEK-transformed cells. Data are shown

as gene expression fold change (log 2) 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 means ± SEM, n=3.

(D) Immunoblot analysis of indicated proteins.

(E) 318 upregulated and 350 downregulated genes show significant 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 (c).

(G) qRT-PCR analysis of indicated genes in HEK-PDK1 cells treated with 10 nM

BI2536 at indicated times.


Figure 7 BI2356 synergizes with BEZ235 to induce synthetic lethality in CRC both in vitro and in vivo (A) Immunoblot analysis of DLD1 cells treated with 100 nM Rapamycin or 100 nM

BEZ235 for 48 hr.

(B) Immunoblot analysis of DLD1, SW480 and HT15 cells treated with 10 nM BI2536,

100 nM BEZ235 alone or combination for 48 hr.

(C) Sub-G1 detection of apoptosis in DLD1, SW480 and HT15 cells treated as (B).

(D) The growth curves of DLD1, SW480 and HT15 cells treated with 10 nM BI2536,

100 nM BEZ235 single or combination for 4 days. RLU means relative luminescence

units.

(E) Xenograft tumor growth of SW480 and HT15 cells in nude mice treated with BI2536

at 50 mg/kg or BEZ235 at 35 mg/kg or both, every other day as described in

Experimental Procedures. Error bars represent ± SEM (n=6 per group).





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Published OnlineFirst July 25, 2013.Cancer Discovery Jing Tan, Zhimei Li, Puay Leng Lee, et al. and Resistance to mTOR-targeted TherapyOncogenic Transformation and Tumor Initiating Cell Activation PDK1 Signaling Towards PLK1-Myc Activation Confers

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PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumor-initiating cell...

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