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PTEN-Deficient Tumors Depend on AKT2 for Maintenance and Survival

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PTEN-Deficient Tumors Depend on AKT2 for Maintenance and Survival Published OnlineFirst May 16, 2014; DOI: 10.1158/2159-8290.CD-13-0873 RESEARCH ARTICLE PTEN-Defi cient Tumors Depend on AKT2 for Maintenance and Survival Y. Rebecca Chin 1 , Xin Yuan 2 , Steven P. Balk 2 , and Alex Toker 1 Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 16, 2014; DOI: 10.1158/2159-8290.CD-13-0873 ABSTRACT Loss of PTEN is a common event in many cancers and leads to hyperactivation of the PI3K–AKT signaling pathway. The mechanisms by which AKT isoforms medi- ate signaling to phenotypes associated with PTEN inactivation in cancer have not been defi ned. Here, we show that AKT2 is exclusively required for PTEN-defi cient prostate tumor spheroid maintenance, whereas AKT1 is dispensable. shRNA silencing of AKT2 but not AKT1 promotes regression of prostate cancer xenografts. Mechanistically, we show that AKT2 silencing upregulates p21 and the proapop- totic protein BAX and downregulates the insulin-like growth factor receptor-1. We also show that p21 is an effector of AKT2 in mediating prostate tumor maintenance. Moreover, AKT2 is also exclusively required for the maintenance and survival of other PTEN-defi cient solid tumors, including breast cancer and glioblastoma. These fi ndings identify a specifi c function for AKT2 in mediating survival of PTEN-defi cient tumors and provide a rationale for developing therapeutics targeting AKT2. SIGNIFICANCE: Depletion of AKT2, but not AKT1, induces potent tumor regression in PTEN-defi cient prostate cancer xenografts, concomitant with upregulation of p21, which may serve as a potential biomarker for screening AKT2 activity in clinical samples. The specifi c role of AKT2 in tumor mainte- nance provides a rationale for the development of isoform-specifi c inhibitors for patients with PTEN- defi cient cancers. Cancer Discov; 4(8); 942–55. ©2014 AACR. INTRODUCTION stream targets of PI3K and PTEN may provide more viable therapeutic strategies. The PI3K signaling pathway is frequently deregulated in The best-understood effector activated downstream of PI3K virtually all human solid tumors ( 1 ). Upon activation by is the protein kinase AKT, which encompasses three isoforms, growth factors, class IA PI3K phosphorylates phosphatidyli- AKT1, AKT2, and AKT3 ( 8, 9 ). PIP binds the pleckstrin nositol-4,5-bisphosphate (PIP ) at the plasma membrane 3 2 homology domain of AKT, effectively recruiting it to the to generate phosphatidylinositol-3,4,5-trisphosphate (PIP ; 3 plasma membrane, where it is activated by phosphorylation ref. 2 ). PIP fulfi lls an essential second messenger role by 3 at threonine 308 and serine 473 by PDK1 and the mTOR recruiting inactive signaling proteins to the plasma mem- complex 2 (mTORC2), respectively ( 10–12 ). Activated AKT brane, resulting in the activation of numerous pathways that then translocates to distinct subcellular compartments where transduce the signal to a plethora of cellular processes ( 3 ). it phosphorylates numerous substrates, many of which are The intracellular levels of PIP are tightly regulated by the 3 oncogenes or tumor suppressors ( 13 ). The essential role of opposing activities of PI3K and PTEN, a PIP 3 ′-phosphatase 3 AKT in tumorigenesis has led to the development of a number that dephosphorylates PIP back to PIP ( 4, 5 ). The p110α 3 2 of fi rst-generation pan-AKT inhibitors currently in clinical tri- catalytic subunit of PI3K, encoded by the PIK3CA gene, is als ( 14 ). Although the three AKT isoforms share a high degree frequently activated by somatic mutation in many epithelial of sequence identity and are regulated by similar mechanisms, cancers, including breast, endometrial, and colon cancers ( 3 ). studies have highlighted distinct functions of AKT isoforms in In contrast, PIK3CA mutations are rare in highly aggressive cancer progression (reviewed in ref. 15 ). For example, whereas metastatic prostate cancer. Instead, loss of PTEN due to LOH AKT2 promotes breast cancer cell migration and metastatic or inactivating mutations is the predominant mechanism dissemination, AKT1 can actually function as a metastasis driving PI3K pathway activation in prostate tumors ( 6, 7 ). suppressor ( 9 , 16–19 ). These and other studies suggest that The critical role of the PI3K pathway in tumorigenesis has led AKT isoform–selective inhibitors might provide more optimal to the development of numerous small-molecule inhibitors therapeutic responses in tumor-specifi c contexts. targeting PI3K ( 1 ). However , as a tumor suppressor, PTEN A critical role for AKT in PTEN-defi cient tumors is evident has yet to be targeted therapeutically, and to this end, down- from a number of studies. PTEN heterozygous mice develop tumors spontaneously in multiple organs, concomitant with hyperphosphorylated AKT ( 20–22 ). Prostate tumor develop- Authors’ Affi liations: 1 Department of Pathology and 2 Hematology-Oncology ment induced by PTEN loss requires functional mTORC2 Division, Department of Medicine, Beth Israel Deaconess Medical Center, ( 23 ). Similarly, mice lacking AKT1 are protected from tum- Harvard Medical School, Boston, Massachusetts origenesis induced by PTEN loss ( 24 ). Curiously, a more recent Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). study indicated that inactivation of AKT2 has little or no con- sequence on prostate neoplasia, explained in part by the rela- Corresponding Author: Alex Toker, Department of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, EC/CLS633A, Boston, tively minimal impact of AKT2 loss on total AKT activity and MA 02215. Phone: 617-735-2482; Fax: 617-735-2480; E-mail: atoker@ also an increase in blood insulin levels ( 25 ). In contrast, AKT2 bidmc.harvard.edu is required for proliferation and invasive migration of PTEN- doi: 10.1158/2159-8290.CD-13-0873 defi cient glioblastoma ( 26, 27 ). In late-stage colorectal cancer, © 2014 American Association for Cancer Research. AKT2 is highly expressed and functions synergistically with AUGUST 2014CANCER DISCOVERY | 943 Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 16, 2014; DOI: 10.1158/2159-8290.CD-13-0873 RESEARCH ARTICLE Chin et al. − + A B Dox Dox 10,000 8,000 Figure 1. AKT2 silencing induces disin- 6,000 tegration of prostate tumor spheroids. A, AKT1 AKT2 4,000 LNCaP cells expressing tet-on AKT isoform Tet-on shRNA v Tet-on 2,000 (pixel area) shRNA or vector control (v) were treated −−+ −+ CTL vector Spheroid size 0 Dox Dox −+ with doxycycline (dox; 100 ng/mL) for 2 AKT1 days. Whole-cell lysates were subjected to 10,000 immunoblotting. B, LNCaP cells containing 8,000 AKT2 Tet-on 6,000 tet-on AKT1 or AKT2 shRNA were grown AKT1 4,000 in 3D culture for 14 days in the presence shRNA 2,000 or absence of doxycycline. Representative p85 (pixel area) *** Spheroid size 0 Dox −+ phase-contrast images are shown. Spheroid size was quantifi ed in pixel area using ImageJ 10,000 and depicted in the bar graph. Error bars, Tet-on 8,000 mean ± SEM; ***, P < 0.001 (Student t test; AKT2 6,000 = shRNA 4,000 n 30). C, LNCaP cells were cultured in 3D 2,000 for 7 or 13 days, followed by doxycycline (pixel area) *** Spheroid size 0 treatment for 10 or 4 days. ***, P < 0.001 Dox − + (Student t test, n = 30). Spheroid size was Dox quantifi ed in pixel area and depicted in the bar graph. Error bars, mean ± SEM; ***, P < 0.001 (Student t test; n = 30). Results are Day 0714 representative of at least three independent experiments. C − 14,000 Dox Dox 4 d Dox 10 d 12,000 10,000 8,000 6,000 Tet-on 4,000 (pixel area) 2,000 CTL vector Spheroid size 0 Dox (d)− 10 10,000 8,000 Tet-on 6,000 AKT1 shRNA 4,000 2,000 (pixel area) Spheroid size 0 Dox (d) − 10 10,000 Tet-on 8,000 AKT2 shRNA 6,000 4,000 2,000 *** (pixel area) Spheroid size 0 Dox (d) − 10 Dox Dox Day 0 7 13 17 PTEN loss to promote metastasis ( 28 ). It has also been dem- RESULTS onstrated that deletion of AKT2 in PTEN -null mice attenuates AKT2 Silencing Induces Prostate Tumor hepatic injury, thereby delaying liver tumor development ( 29 ). Spheroid Disintegration Although the contribution of AKT in tumor initiation in the context of PTEN inactivation has been determined, the The role of AKT in the initiation of PTEN-defi cient pros- role of AKT isoforms in the maintenance of established PTEN- tate tumors has been studied ( 24, 25 ). However, the function null tumors is unknown. Moreover, it is unclear whether of AKT isoforms in modulating cancer cell survival in this PTEN-defi cient tumors depend on specifi c AKT isoform(s) context has not been determined. To assess the roles of AKT for survival signaling. Here, we have used an inducible shRNA isoforms in tumor initiation as well as maintenance, we gener- strategy to silence individual AKT isoforms in tumor spheroids ated isoform-specifi c shRNAs in a doxycycline (dox)-regulated grown in three-dimensional (3D) culture. We fi nd that in system. Because of the high frequency of PTEN inactivation PTEN-defi cient tumor cells depletion of AKT2 induces apop- (up to 50%) observed in human prostate cancers ( 6 ), we fi rst tosis and leads to regression of established prostate xenografts. introduced shRNAs in the PTEN-defi cient prostate LNCaP In contrast, downregulation of AKT1 or AKT3 has no effect line. Addition of doxycycline to LNCaP cells results in highly on the integrity of tumor spheroids. We also demonstrate that selective silencing of AKT1 and AKT2 (LNCaP cells do not p21 is a downstream effector of AKT2 in modulating tumor- express AKT3; Fig. 1A ). We next performed 3D spheroid cell survival. These data identify AKT2 as the critical isoform assays with Matrigel, because growth of tumor cells in 3D for driving maintenance of established PTEN-defi cient cancers.
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