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Upregulation of AKT3, but not AKT1 or AKT2, in AKT inhibitor-resistant MDA-MB-468 breast tumor cells.

PDF file 1017K, Figure S1 shows the functions of Akt1 and Akt2 in the proliferation and migration of prostate tumor cells. Figure S2 demonstrates that all three Akt isoforms play a critical role in MDA-MB-468 breast cancer cell proliferation and formation of tumor spheroids. Figure S3 shows that Akt2 does not play a critical role in the survival of tumor cells with wild-type PTEN. Figure S4 shows the expression and phosphorylation levels of Akt1 and Akt2 in prostate tumor cells. Figure S5 demonstrates a critical role of p21 in Akt2-mediated cell cycle progression. Figure S6 shows that Bax does not play a role in Akt2-mediated spheroid survival. Figure S7 shows the volume of individual tumors measured in the xenograft studies for Fig. 6A

Knockdown of AKT1 or AKT2 does not sensitizes AKTi-resistant breast tumor cells to MK2206.

Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor–resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor–resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964–74. ©2016 AACR.

Knockdown of AKT3 sensitizes AKTi-resistant breast tumor cells to MK2206.

Figure S1. The Sin1-PH domain binds the mTOR kinase domain to inhibit mTOR kinase activity, Related to Figure 1. Figure S2. The PH domain is a unique and physiological functional PH domain, Related to Figure 2. Figure S3. PI(3,4,5)P3 binds the Sin1-PH motif to activate mTORC2, Related to Figure 3. Figure S4. The presence of PIP3 species leads to attenuated Sin1-PH domain interaction with mTOR-KD, Related to Figure 4. Figure S5. The Sin1-PH motif mainly interacts with PI(3,4,5)P3 through three critical residues including R393, K428 and K464, Related to Figure 5. Figure S6. The cancerous Sin1-PH mutant lose binding with the mTOR-kinase domain, leading to elevated Akt-S473 phosphorylation and enhanced oncogenic activities, Related to Figure 6. Figure S7. C-terminal tagging of KRas-CAAX to Sin1 could partially rescue the deficiency of the Sin1-CAA mutant in membrane recruitment and mTORC2 activation, Related to Figure 7.

Table showing differentially expressed genes in T47D MK2206-resistant cells.

PDF file - 219K, Effects of RhoB deletion on MT-myc induced mammary tumorigenesis in virgin female mice

PDF file - 22K

PDF file - 54K, RhoB is downregulated in MCF7 cells by antisense oligonucleotides

PDF file - 21K, RhoB is downregulated in MDA-MB-231 cells using lentivirus shRNA

Supplementary Information, Methods, Table 1, Figures 1-7 from 3-Phosphoinositide–Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma

PDF file - 34K, Level of expression of mRNA of different genes in RhoB+/+ and RhoB-/- mouse tumor cells

Triple-negative breast cancer (TNBC) is currently the only major breast tumor subtype without effective targeted therapy and, as a consequence, in general has a poor outcome. To identify new therapeutic targets in TNBC, we performed a short hairpin RNA (shRNA) screen for protein kinases commonly amplified and overexpressed in breast cancer. Using this approach, we identified AKT3 as a gene preferentially required for the growth of TNBCs. Downregulation of Akt3 significantly inhibits the growth of TNBC lines in three-dimensional (3D) spheroid cultures and in mouse xenograft models, whereas loss of Akt1 or Akt2 have more modest effects. Akt3 silencing markedly upregulates the p27 cell-cycle inhibitor and this is critical for the ability of Akt3 to inhibit spheroid growth. In contrast with Akt1, Akt3 silencing results in only a minor enhancement of migration and does not promote invasion. Depletion of Akt3 in TNBC sensitizes cells to the pan-Akt inhibitor GSK690693. These results imply that Akt3 has a specific function in TNBCs; thus, its therapeutic targeting may provide a new treatment option for this tumor subtype. Cancer Res; 74(3); 964–73. ©2013 AACR.

PDF file - 52K, Level of silencing of Akt in RhoB-/- and RhoB+/+ mouse cells

PDF file - 26K, Level of EGFR on the surface of human MDA-MB-231 cells

PDF file - 164K, AKT3 Expression and Survival in TNBC.

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

Background Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis. By performing multiomic profiling, we recently uncovered super-enhancer heterogeneity between breast cancer subtypes. Our data also revealed TCOF1 as a putative TNBC-specific super-enhancer-regulated gene. TCOF1 plays a critical role in craniofacial development but its function in cancer remains unclear. Methods Overall survival and multivariant Cox regression analyses were conducted using the METABRIC data set. The effect of TCOF1 knockout on TNBC growth and stemness was evaluated by in vitro and in vivo assays. RNA-seq and rescue experiments were performed to explore the underlying mechanisms. Results TCOF1 is frequently upregulated in TNBC and its elevated expression correlates with shorter overall survival. TCOF1 depletion significantly inhibits the growth and stemness of basal-like TNBC, but not of mesenchymal-like cells, highlighting the distinct molecular dependency in different TNBC subgroups. RNA-seq uncovers several stem cell molecules regulated by TCOF1. We further demonstrate that KIT is a downstream effector of TCOF1 in mediating TNBC stemness. TCOF1 expression in TNBC is regulated by the predicted super-enhancer. Conclusions TCOF1 depletion potently attenuates the growth and stemness of basal-like TNBC. Expression of TCOF1 may serve as a TNBC prognostic marker and a therapeutic target.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor–resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor–resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964–74. ©2016 AACR.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology. Cancer Res; 73(1); 50–61. ©2012 AACR.

Summary Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.

Invasive migration of carcinoma cells is a prerequisite for the metastatic dissemination of solid tumours. Numerous mechanisms control the ability of cancer cells to acquire a motile and invasive phenotype, and subsequently degrade and invade the basement membrane. Several genes that are up-regulated in breast carcinoma are responsible for mediating the metastatic cascade. Recent studies have revealed that the NFAT (nuclear factor of activated T-cells) is a transcription factor that is highly expressed in aggressive breast cancer cells and tissues, and mediates invasion through transcriptional induction of pro-invasion and migration genes. In the present paper we demonstrate that NFAT promotes breast carcinoma invasion through induction of GPC (glypican) 6, a cell-surface glycoprotein. NFAT transcriptionally regulates GPC6 induction in breast cancer cells and binds to three regulatory elements in the GPC6 proximal promoter. Expression of GPC6 in response to NFAT signalling promotes invasive migration, whereas GPC6 silencing with shRNA (small-hairpin RNA) potently blocks this phenotype. The mechanism by which GPC6 promotes invasive migration involves inhibition of canonical β-catenin and Wnt signalling, and up-regulation of non-canonical Wnt5A signalling leading to the activation of JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Thus GPC6 is a novel NFAT target gene in breast cancer cells that promotes invasive migration through Wnt5A signalling.

Numerous studies have shown that Akt isoforms promote tumorigenesis by enhancing cancer cell survival and growth, and it is well established that signaling through the Akt upstream regulator PI 3-K enhances cancer cell migration. Therefore, it is conventionally accepted that PI 3-K/Akt pathway promotes tumor formation and metastasis. A few years ago, studies from several laboratories added a new layer to the pleiotropic effects of Akt function by showing that the Akt1 isoform inhibits breast cancer cell migration and invasion, whereas Akt2 promotes these phenotypes. These studies challenged the dogma and identified non-redundant functions of Akt isoforms in cancer progression. The identification of palladin as an Akt1-specific substrate in our recently published work has exemplified distinct Akt isoform-specific signaling in breast cancer. Here, we review these findings and discuss the implications for the understanding of the mechanistic basis for designing more effective anti-cancer therapeutics targeting the Akt pathway.

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell cycle progression. However, it remains largely elusive whether hyper-phosphorylated, non-E2F1-interacting form, of Rb has any physiological role. Here, we report that hyper-phosphorylated Rb directly binds to, and suppresses the function of mTORC2, but not mTORC1. Mechanistically, Rb, but not p107 nor p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D, or using CDK4/6 inhibitors, releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer.

The receptor internalization and degradation (RID) complex of adenovirus plays an important role in modulating the immune response by downregulating the surface levels of tumour necrosis factor receptor 1 (TNFR1), thereby inhibiting NF-κB activation. Total cellular content of TNFR1 is also reduced in the presence of RID, which can be inhibited by treatment with lysosomotropic agents. In this report, surface biotinylation experiments revealed that, although RID and TNFR1 were able to form a complex on the cell surface, the rate of TNFR1 endocytosis was not affected by RID. However, the degradation of internalized TNFR1 was enhanced significantly in the presence of RID. Therefore, these data suggest that RID downregulates TNFR1 levels by altering the fate of internalized TNFR1 that becomes associated with RID at the plasma membrane, probably by promoting its sorting into endosomal/lysosomal degradation compartments.

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 mediate signaling to phenotypes associated with PTEN inactivation in cancer have not been defined. Here, we show that AKT2 is exclusively required for PTEN-deficient 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 proapoptotic 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-deficient solid tumors, including breast cancer and glioblastoma. These findings identify a specific function for AKT2 in mediating survival of PTEN-deficient tumors and provide a rationale for developing therapeutics targeting AKT2. Significance: Depletion of AKT2, but not AKT1, induces potent tumor regression in PTEN-deficient prostate cancer xenografts, concomitant with upregulation of p21, which may serve as a potential biomarker for screening AKT2 activity in clinical samples. The specific role of AKT2 in tumor maintenance provides a rationale for the development of isoform-specific inhibitors for patients with PTEN-deficient cancers. Cancer Discov; 4(8); 942–55. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 855

Akt activation is a hallmark of human cancers. Here, we report a critical mechanism for regulation of Akt activity by the splicing kinase SRPK1, a downstream Akt target for transducing growth signals to regulate splicing. Surprisingly, we find that SRPK1 has a tumor suppressor function because ablation of SRPK1 in mouse embryonic fibroblasts induces cell transformation. We link the phenotype to constitutive Akt activation from genome-wide phosphoproteomics analysis and discover that downregulated SRPK1 impairs the recruitment of the Akt phosphatase PHLPP1 (pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase) to Akt. Interestingly, SRPK1 overexpression is also tumorigenic because excess SRPK1 squelches PHLPP1. Thus, aberrant SRPK1 expression in either direction induces constitutive Akt activation, providing a mechanistic basis for previous observations that SRPK1 is downregulated in some cancer contexts and upregulated in others.

Triple-negative breast cancer (TNBC) is currently the only major breast tumor subtype without effective targeted therapy and, as a consequence, in general has a poor outcome. To identify new therapeutic targets in TNBC, we performed a short hairpin RNA (shRNA) screen for protein kinases commonly amplified and overexpressed in breast cancer. Using this approach, we identified AKT3 as a gene preferentially required for the growth of TNBCs. Downregulation of Akt3 significantly inhibits the growth of TNBC lines in three-dimensional (3D) spheroid cultures and in mouse xenograft models, whereas loss of Akt1 or Akt2 have more modest effects. Akt3 silencing markedly upregulates the p27 cell-cycle inhibitor and this is critical for the ability of Akt3 to inhibit spheroid growth. In contrast with Akt1, Akt3 silencing results in only a minor enhancement of migration and does not promote invasion. Depletion of Akt3 in TNBC sensitizes cells to the pan-Akt inhibitor GSK690693. These results imply that Akt3 has a specific function in TNBCs; thus, its therapeutic targeting may provide a new treatment option for this tumor subtype. Cancer Res; 74(3); 964–73. ©2013 AACR.

mTOR serves as a central regulator of cell growth and metabolism by forming two distinct complexes, mTORC1 and mTORC2. Although mechanisms of mTORC1 activation by growth factors and amino acids have been extensively studied, the upstream regulatory mechanisms leading to mTORC2 activation remain largely elusive. Here, we report that the pleckstrin homology (PH) domain of SIN1, an essential and unique component of mTORC2, interacts with the mTOR kinase domain to suppress mTOR activity. More importantly, PtdIns(3,4,5)P3, but not other PtdInsPn species, interacts with SIN1-PH to release its inhibition on the mTOR kinase domain, thereby triggering mTORC2 activation. Mutating critical SIN1 residues that mediate PtdIns(3,4,5)P3 interaction inactivates mTORC2, whereas mTORC2 activity is pathologically increased by patient-derived mutations in the SIN1-PH domain, promoting cell growth and tumor formation. Together, our study unravels a PI3K-dependent mechanism for mTORC2 activation, allowing mTORC2 to activate AKT in a manner that is regulated temporally and spatially by PtdIns(3,4,5)P3. Significance: The SIN1-PH domain interacts with the mTOR kinase domain to suppress mTOR activity, and PtdIns(3,4,5)P3 binds the SIN1-PH domain to release its inhibition on the mTOR kinase domain, leading to mTORC2 activation. Cancer patient–derived SIN1-PH domain mutations gain oncogenicity by loss of suppressing mTOR activity as a means to facilitate tumorigenesis. Cancer Discov; 5(11); 1194–209. ©2015 AACR. See related commentary by Yuan and Guan, p. 1127. This article is highlighted in the In This Issue feature, p. 1111

Summary The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCF βTrCP . DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds βTrCP. Failure to degrade DEPTOR through either degron mutation or βTrCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently deregulated in cancer. Downstream of PI3K, Akt1 and Akt2 have opposing roles in breast cancer invasive migration, leading to metastatic dissemination. Here, we identify palladin, an actin-associated protein, as an Akt1-specific substrate that modulates breast cancer cell invasive migration. Akt1, but not Akt2, phosphorylates palladin at Ser507 in a domain that is critical for F-actin bundling. Downregulation of palladin enhances migration and invasion of breast cancer cells and induces abnormal branching morphogenesis in 3D cultures. Palladin phosphorylation at Ser507 is required for Akt1-mediated inhibition of breast cancer cell migration and also for F-actin bundling, leading to the maintenance of an organized actin cytoskeleton. These findings identify palladin as an Akt1-specific substrate that regulates cell motility and provide a molecular mechanism that accounts for the functional distinction between Akt isoforms in breast cancer cell signaling to cell migration.

The phosphoinositide 3-kinase (PI3K) pathway regulates a multitude of cellular processes. Deregulation of PI3K signaling is often observed in human cancers. A major effector of PI3K is Akt/protein kinase B (PKB). Recent studies have pointed to distinct roles of Akt/PKB isoforms in cancer cell signaling. Studies have shown that Akt1 (PKBα) can attenuate breast cancer cell motility, whereas Akt2 (PKBβ) enhances this phenotype. Here, we have evaluated the mechanism by which Akt1 blocks the migration of breast cancer cells through the transcription factor NFAT. A major effector of Akt/PKB is glycogen synthase kinase-3β (GSK-3β), also a NFAT kinase. Inhibition of GSK-3β using short hairpin RNA or a selective inhibitor potently blocks breast cancer cell migration concomitant with a reduction in NFAT activity. GSK-3β-mediated inhibition of NFAT activity is due to proteasomal degradation. Experiments using GSK-3β mutants, which are unresponsive to Akt/PKB, reveal that inhibition of cell migration by Akt/PKB is mediated by GSK-3β. These effects are recapitulated at the levels of NFAT degradation by the proteasome. Our studies show that activation of Akt/PKB leads to inactivation of the effector GSK-3β and the outcome of this signaling event is degradation of NFAT by the proteasome and subsequent inhibition of cell migration. (Mol Cancer Res 2009;7(3):425–32)

The serine/threonine protein kinase Akt is a major signal transducer of the phosphoinositide 3-kinase (PI 3-K) pathway in all cells and tissues and plays a pivotal role in the maintenance of cellular processes including cell growth, proliferation, survival and metabolism. The frequent aberrant activation of the PI 3-K/Akt pathway in human cancer has made it an attractive therapeutic target. Numerous studies have provided a comprehensive understanding of the specific functions of Akt signaling in cancer cells as well as the surrounding tumor microenvironment and this has informed and enabled the development of therapeutic drugs to target both PI 3-K and Akt. However, recent studies have provided evidence for distinct functions of the three mammalian Akt isoforms, particularly with respect to the regulation of cell motility and metastasis of breast cancer. Here we discuss the mechanisms by which Akt signaling contributes to invasive migration and tumor metastasis, and highlight recent advances in our understanding of the contribution of the Akt pathway in the tumor-associated stroma.

Resistance to targeted therapy poses a major challenge for successful treatment of cancers. Despite a number of drugs targeting the PI 3-K/Akt pathway are in clinical development, mechanisms of resistance to drugs targeting this pathway have not been studied extensively. To determine how breast cancer cells develop resistance to Akt inhibitors (Akti), we generated Akti-resistant lines using dose escalation regimens with pan-Akt inhibitors (ATP-competitive: GDC0068, GSK690693; Allosteric: MK2206). Interestingly, in multiple Akti-resistant breast cancer lines, we observed a robust increase in the protein expression of Akt3, but not Akt1 or Akt2. We were intrigued by this observation, as we have recently demonstrated that Akt3 has a predominant function in regulating growth of triple negative breast cancer cells in three-dimensional spheroids and in xenograft models. The increased expression of Akt3 in Akti-resistant cells occurs via a genetic event, since the levels of Akt3 remain elevated after 3 weeks of Akt inhibitor removal. Moreover, upon Akt inhibitor removal in resistant lines, cells remain resistant to Akt inhibition. The enhanced Akt3 expression is observed in both luminal and basal-like triple-negative breast cancer resistant lines. Importantly, knockdown of Akt3, but not Akt1 or Akt2, results in re-sensitization of resistant cells to Akti. Mechanistically, using anti-phosphotyrosine receptor antibody array (RTK array), we observed increased phosphorylation of several RTKs in resistant cells, including EGFR, Her2, and ROR1. Akt3 knockdown in resistant cells results in reduction of pEGFR, pHer3 and pHer2, suggesting that Akt3 plays a role in the regulation of multiple RTKs in resistant cells. Epithelial-mesenchymal transition (EMT) is implicated in therapeutic resistance of cancers. Interestingly, several of our resistant lines display EMT properties including downregulation of E-Cadherin, and upregulation of N-Cadherin as well as Vimentin. We propose that enhanced Akt3 expression is a general mechanism by which breast cancer cells acquire resistance to Akti targeted therapy. Current efforts are aimed at investigating the mechanistic basis for Akt3-mediated resistance, and demonstrating the physiological relevance of these findings in clinical samples as well as in mouse models. The role of Akt3 in regulating EMT during establishment of resistance will also be explored. These studies will provide insights into molecular mechanisms of therapeutic resistance to drugs targeting the PI 3-K/Akt pathway, as well as information for designing combination strategies and next generation therapy. Citation Format: Y Rebecca Chin, Alex Toker. Increased Akt3 expression as a resistance mechanism to targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A42.

Proteins encoded in adenovirus early region 3 have important immunoregulatory properties. We have recently shown that the E3-10.4K/14.5K (RIDα/β) complex downregulates tumor necrosis factor receptor 1 (TNFR1) expression at the plasma membrane. To study the role of the RIDβ tyrosine sorting motif in the removal of surface TNFR1, tyrosine 122 on RIDβ was mutated to alanine or phenylalanine. Both RIDβ mutations not only abolished the downregulation of surface TNFR1 but paradoxically increased surface TNFR1 levels. RID also downregulates other death receptors, such as FAS; however, surface FAS expression was not increased by RIDβ mutants, suggesting that regulation of TNFR1 and that of FAS by RID are mechanistically different. In the mixing experiments, the wild-type (WT) RID-mediated TNFR1 downregulation was partially inhibited in the presence of RIDβ mutants, indicating that the mutants compete for TNFR1 access. Indeed, an association between RIDβ and TNFR1 was shown by coimmunoprecipitation. In contrast, the mutants did not affect the WT RID-induced downregulation of FAS. These differential effects support a model in which RID associates with TNFR1 on the plasma membrane, whereas RID probably associates with FAS in a cytoplasmic compartment. By using small interfering RNA against the μ2 subunit of adaptor protein 2, dominant negative dynamin construct K44A, and the lysosomotropic agents bafilomycin A1 and ammonium chloride, we also demonstrated that surface TNFR1 was internalized by RID by a clathrin-dependent process involving μ2 and dynamin, followed by degradation of TNFR1 via an endosomal/lysosomal pathway.

The phosphatidylinositol 3-kinase/Akt pathway is responsible for key aspects of tumor progression, and is frequently hyperactivated in cancer. We have recently identified palladin, an actin-bundling protein that functions to control the actin cytoskeleton, as an Akt1-specific substrate that inhibits breast cancer cell migration. Here we have identified a role for Akt isoforms in the regulation of palladin expression. Akt2, but not Akt1, enhances palladin expression by maintaining protein stability and upregulating transcription. These data reveal that Akt signaling regulates the stability of palladin, and further supports the notion that Akt isoforms have distinct and specific roles in tumorigenesis.