Publications by authors named "Alex Toker"

87 Publications

Lactate Lights up PI3K Inhibitor Resistance in Breast Cancer.

Cancer Cell 2020 10;38(4):441-443

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

Predictive biomarkers can facilitate optimal patient selection for targeted cancer therapies. In this issue of Cancer Cell, Ros et al. show the utility of noninvasive metabolic imaging of labeled carbon transfer from pyruvate to lactate to detect early response and FOXM1-mediated resistance to PI3K inhibition in estrogen-receptor-positive breast cancer.
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http://dx.doi.org/10.1016/j.ccell.2020.09.011DOI Listing
October 2020

Metabolic pathway alterations in microvascular endothelial cells in response to hypoxia.

PLoS One 2020 9;15(7):e0232072. Epub 2020 Jul 9.

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

The vasculature within a tumor is highly disordered both structurally and functionally. Endothelial cells that comprise the vasculature are poorly connected causing vessel leakage and exposing the endothelium to a hypoxic microenvironment. Therefore, most anti-angiogenic therapies are generally inefficient and result in acquired resistance to increased hypoxia due to elimination of the vasculature. Recent studies have explored the efficacy of targeting metabolic pathways in tumor cells in combination with anti-angiogenic therapy. However, the metabolic alterations of endothelial cells in response to hypoxia have been relatively unexplored. Here, we measured polar metabolite levels in microvascular endothelial cells exposed to short- and long-term hypoxia with the goal of identifying metabolic vulnerabilities that can be targeted to normalize tumor vasculature and improve drug delivery. We found that many amino acid-related metabolites were altered by hypoxia exposure, especially within alanine-aspartate-glutamate, serine-threonine, and cysteine-methionine metabolism. Additionally, there were significant changes in de novo pyrimidine synthesis as well as glutathione and taurine metabolism. These results provide key insights into the metabolic alterations that occur in endothelial cells in response to hypoxia, which serve as a foundation for future studies to develop therapies that lead to vessel normalization and more efficient drug delivery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232072PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347218PMC
September 2020

The INPP4B Tumor Suppressor Modulates EGFR Trafficking and Promotes Triple-Negative Breast Cancer.

Cancer Discov 2020 Aug 8;10(8):1226-1239. Epub 2020 Jun 8.

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Inactivation of the tumor suppressor lipid phosphatase INPP4B is common in triple-negative breast cancer (TNBC). We generated a genetically engineered TNBC mouse model deficient in . We found a dose-dependent increase in tumor incidence in homozygous and heterozygous knockout mice compared with wild-type (WT), supporting a role for as a tumor suppressor in TNBC. Tumors derived from knockout mice are enriched for AKT and MEK gene signatures. Consequently, mice with deficiency are more sensitive to PI3K or MEK inhibitors compared with WT mice. Mechanistically, we found that deficiency increases PI(3,4)P levels in endocytic vesicles but not at the plasma membrane. Moreover, loss delays degradation of EGFR and MET, while promoting recycling of receptor tyrosine kinases (RTK), thus enhancing the duration and amplitude of signaling output upon growth factor stimulation. Therefore, inactivation in TNBC promotes tumorigenesis by modulating RTK recycling and signaling duration. SIGNIFICANCE: Inactivation of the lipid phosphatase is frequent in TNBC. Using a genetically engineered mouse model, we show that INPP4B functions as a tumor suppressor in TNBC. INPP4B regulates RTK trafficking and degradation, such that loss of INPP4B prolongs both PI3K and ERK activation..
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http://dx.doi.org/10.1158/2159-8290.CD-19-1262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415683PMC
August 2020

Inhibition of the polyamine synthesis enzyme ornithine decarboxylase sensitizes triple-negative breast cancer cells to cytotoxic chemotherapy.

J Biol Chem 2020 05 5;295(19):6263-6277. Epub 2020 Mar 5.

Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.
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http://dx.doi.org/10.1074/jbc.RA119.012376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212655PMC
May 2020

Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling.

Cell Chem Biol 2020 01 16;27(1):66-73.e7. Epub 2019 Dec 16.

Department of Pathology, Medicine and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small-molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of the ATP-competitive AKT inhibitor GDC-0068 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 h, even after compound washout. Our findings suggest that AKT degradation may confer prolonged pharmacological effects compared with inhibition, and highlight the potential advantages of AKT-targeted degradation.
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http://dx.doi.org/10.1016/j.chembiol.2019.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980747PMC
January 2020

Double trouble for cancer gene.

Authors:
Alex Toker

Science 2019 11;366(6466):685-686

Beth Israel Deaconess Medical Center, Department of Pathology, Harvard University Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1126/science.aaz4016DOI Listing
November 2019

AKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions.

Nat Cell Biol 2019 02 28;21(2):226-237. Epub 2019 Jan 28.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Aberrant activation of AKT disturbs the proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand the upstream signalling pathways governing AKT activation. Here, we report that AKT undergoes SETDB1-mediated lysine methylation to promote its activation, which is antagonized by the Jumonji-family demethylase KDM4B. Notably, compared with wild-type mice, mice harbouring non-methylated mutant Akt1 not only exhibited reduced body size but were also less prone to carcinogen-induced skin tumours, in part due to reduced AKT activation. Mechanistically, the interaction of phosphatidylinositol (3,4,5)-trisphosphate with AKT facilitates its interaction with SETDB1 for subsequent AKT methylation, which in turn sustains AKT phosphorylation. Pathologically, genetic alterations, including SETDB1 amplification, aberrantly promote AKT methylation to facilitate its activation and oncogenic functions. Thus, AKT methylation is an important step, synergizing with PI3K signalling to control AKT activation. This suggests that targeting SETDB1 signalling could be a potential therapeutic strategy for combatting hyperactive AKT-driven cancers.
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http://dx.doi.org/10.1038/s41556-018-0261-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377565PMC
February 2019

PI 3-Kinase Signaling: AKTing up inside the Cell.

Mol Cell 2018 09;71(6):875-876

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

In this issue of Molecular Cell, Liu et al. (2018) show that PI34P2 and PIP3, the lipid products of class I phosphoinositide 3-kinase (PI3K), display distinct spatiotemporal kinetics in cells that result in differential activation of the effectors AKT1, AKT2, and AKT3.
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http://dx.doi.org/10.1016/j.molcel.2018.09.006DOI Listing
September 2018

Skp2-dependent reactivation of AKT drives resistance to PI3K inhibitors.

Sci Signal 2018 03 13;11(521). Epub 2018 Mar 13.

Department of Pathology, Medicine and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

The PI3K-AKT kinase signaling pathway is frequently deregulated in human cancers, particularly breast cancer, where amplification and somatic mutations of occur with high frequency in patients. Numerous small-molecule inhibitors targeting both PI3K and AKT are under clinical evaluation, but dose-limiting toxicities and the emergence of resistance limit therapeutic efficacy. Various resistance mechanisms to PI3K inhibitors have been identified, including de novo mutations, feedback activation of AKT, or cross-talk pathways. We found a previously unknown resistance mechanism to PI3K pathway inhibition that results in AKT rebound activation. In a subset of triple-negative breast cancer cell lines, treatment with a PI3K inhibitor or depletion of expression ultimately promoted AKT reactivation in a manner dependent on the E3 ubiquitin ligase Skp2, the kinases IGF-1R (insulin-like growth factor 1 receptor) and PDK-1 (phosphoinositide-dependent kinase-1), and the cell growth and metabolism-regulating complex mTORC2 (mechanistic target of rapamycin complex 2), but was independent of PI3K activity or PIP production. Resistance to PI3K inhibitors correlated with the increased abundance of Skp2, ubiquitylation of AKT, cell proliferation in culture, and xenograft tumor growth in mice. These findings reveal a ubiquitin signaling feedback mechanism by which PI3K inhibitor resistance may emerge in aggressive breast cancer cells.
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http://dx.doi.org/10.1126/scisignal.aao3810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902813PMC
March 2018

Oncogenic PI3K promotes methionine dependency in breast cancer cells through the cystine-glutamate antiporter xCT.

Sci Signal 2017 Dec 19;10(510). Epub 2017 Dec 19.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

The precursor homocysteine is metabolized either through the methionine cycle to produce methionine or through the transsulfuration pathway to synthesize cysteine. Alternatively, cysteine can be obtained through uptake of its oxidized form, cystine. Many cancer cells exhibit methionine dependency such that their proliferation is impaired in growth media in which methionine is replaced by homocysteine. We showed that oncogenic and decreased expression of , a gene that encodes a cystine transporter also known as xCT, correlated with increased methionine dependency in breast cancer cells. Oncogenic was sufficient to confer methionine dependency to mammary epithelial cells, partly by decreasing cystine uptake through the transcriptional and posttranslational inhibition of xCT. Manipulation of xCT activity altered the proliferation of breast cancer cells in methionine-deficient, homocysteine-containing media, suggesting that it functionally contributed to methionine dependency. We propose that concurrent with decreased cystine uptake through xCT, mutant cells use homocysteine through the transsulfuration pathway to synthesize cysteine. Consequently, less homocysteine is available to produce methionine, contributing to methionine dependency. These results indicate that oncogenic alters methionine and cysteine utilization, partly by inhibiting xCT to contribute to the methionine dependency phenotype in breast cancer cells.
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http://dx.doi.org/10.1126/scisignal.aao6604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808948PMC
December 2017

Identifying and Targeting Sporadic Oncogenic Genetic Aberrations in Mouse Models of Triple-Negative Breast Cancer.

Cancer Discov 2018 03 4;8(3):354-369. Epub 2017 Dec 4.

Meyer Cancer Center, Weill Cornell Medicine, New York, New York.

Triple-negative breast cancers (TNBC) are genetically characterized by aberrations in and a low rate of activating point mutations in common oncogenes, rendering it challenging in applying targeted therapies. We performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify somatic genetic alterations in mouse models of TNBCs driven by loss of alone or in combination with Amplifications or translocations that resulted in elevated oncoprotein expression or oncoprotein-containing fusions, respectively, as well as frameshift mutations of tumor suppressors were identified in approximately 50% of the tumors evaluated. Although the spectrum of sporadic genetic alterations was diverse, the majority had in common the ability to activate the MAPK/PI3K pathways. Importantly, we demonstrated that approved or experimental drugs efficiently induce tumor regression specifically in tumors harboring somatic aberrations of the drug target. Our study suggests that the combination of WES and RNA-seq on human TNBC will lead to the identification of actionable therapeutic targets for precision medicine-guided TNBC treatment. Using combined WES and RNA-seq analyses, we identified sporadic oncogenic events in TNBC mouse models that share the capacity to activate the MAPK and/or PI3K pathways. Our data support a treatment tailored to the genetics of individual tumors that parallels the approaches being investigated in the ongoing NCI-MATCH, My Pathway Trial, and ESMART clinical trials. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5907916PMC
March 2018

AKT/PKB Signaling: Navigating the Network.

Cell 2017 04;169(3):381-405

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, and Ludwig Center at Harvard, Boston, MA 02215, USA. Electronic address:

The Ser and Thr kinase AKT, also known as protein kinase B (PKB), was discovered 25 years ago and has been the focus of tens of thousands of studies in diverse fields of biology and medicine. There have been many advances in our knowledge of the upstream regulatory inputs into AKT, key multifunctional downstream signaling nodes (GSK3, FoxO, mTORC1), which greatly expand the functional repertoire of AKT, and the complex circuitry of this dynamically branching and looping signaling network that is ubiquitous to nearly every cell in our body. Mouse and human genetic studies have also revealed physiological roles for the AKT network in nearly every organ system. Our comprehension of AKT regulation and functions is particularly important given the consequences of AKT dysfunction in diverse pathological settings, including developmental and overgrowth syndromes, cancer, cardiovascular disease, insulin resistance and type 2 diabetes, inflammatory and autoimmune disorders, and neurological disorders. There has also been much progress in developing AKT-selective small molecule inhibitors. Improved understanding of the molecular wiring of the AKT signaling network continues to make an impact that cuts across most disciplines of the biomedical sciences.
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http://dx.doi.org/10.1016/j.cell.2017.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546324PMC
April 2017

Cross-talk between the CK2 and AKT signaling pathways in cancer.

Adv Biol Regul 2017 05 28;64:1-8. Epub 2017 Mar 28.

Cell Signaling Unit, Department of Surgery, Medicine, Dentistry and Morphology, University of Modena and Reggio Emilia, 41124 Modena, Italy. Electronic address:

CK2 and AKT display a high degree of cross-regulation of their respective functions, both directly, through physical interaction and phosphorylation, and indirectly, through an intense cross-talk of key downstream effectors, ultimately leading to sustained AKT activation. Being CK2 and AKT attractive targets for therapeutic intervention, here we would like to emphasize how AKT and CK2 might influence cell fate through their complex isoform-specific and contextual-dependent cross-talk, to the extent that such functional interplay should be considered when devising therapies that target one or both these key signaling kinases.
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http://dx.doi.org/10.1016/j.jbior.2017.03.002DOI Listing
May 2017

PI3K signaling in cancer: beyond AKT.

Curr Opin Cell Biol 2017 04 24;45:62-71. Epub 2017 Mar 24.

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

The phosphoinositide 3-kinase (PI3K) signaling pathway is one of the most frequently altered pathways in human cancer and has a critical role in driving tumor initiation and progression. Although PI3K and its lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP) have been shown to activate multiple downstream signaling proteins, the vast majority of studies have focused on the protein kinase AKT as the dominant effector of PI3K signaling. However, recent studies have demonstrated many contexts under which other PIP-dependent signaling proteins critically contribute to cancer progression, illustrating the importance of understanding AKT-independent signaling downstream of PI3K. Here, we highlight three PI3K-dependent, but AKT-independent, signaling branches that have recently been shown to have important roles in promoting phenotypes associated with malignancy. First, the PDK1-mTORC2-SGK axis can substitute for AKT in survival, migration, and growth signaling and has emerged as a major mechanism of resistance to PI3K and AKT inhibitors. Second, Rac signaling mediates the reorganization of the actin cytoskeleton to regulate cancer cell migration, invasion, and metabolism. Finally, the TEC family kinase BTK has a critical role in B cell function and malignancy and represents a recent example of an effective therapeutic target in cancer. These mechanisms highlight how understanding PI3K-dependent, but AKT-independent, signaling mechanisms that drive cancer progression will be crucial for the development of novel and more effective approaches for targeting the PI3K pathway for therapeutic benefit in cancer.
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http://dx.doi.org/10.1016/j.ceb.2017.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482768PMC
April 2017

Adaptive Reprogramming of Pyrimidine Synthesis Is a Metabolic Vulnerability in Triple-Negative Breast Cancer.

Cancer Discov 2017 04 2;7(4):391-399. Epub 2017 Mar 2.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Chemotherapy resistance is a major barrier to the treatment of triple-negative breast cancer (TNBC), and strategies to circumvent resistance are required. Using and metabolic profiling of TNBC cells, we show that an increase in the abundance of pyrimidine nucleotides occurs in response to chemotherapy exposure. Mechanistically, elevation of pyrimidine nucleotides induced by chemotherapy is dependent on increased activity of the pyrimidine synthesis pathway. Pharmacologic inhibition of pyrimidine synthesis sensitizes TNBC cells to genotoxic chemotherapy agents by exacerbating DNA damage. Moreover, combined treatment with doxorubicin and leflunomide, a clinically approved inhibitor of the pyrimidine synthesis pathway, induces regression of TNBC xenografts. Thus, the increase in pyrimidine nucleotide levels observed following chemotherapy exposure represents a metabolic vulnerability that can be exploited to enhance the efficacy of chemotherapy for the treatment of TNBC. The prognosis for patients with TNBC with residual disease after chemotherapy is poor. We find that chemotherapy agents induce adaptive reprogramming of pyrimidine synthesis and show that this response can be exploited pharmacologically, using clinically approved inhibitors of pyrimidine synthesis, to sensitize TNBC cells to chemotherapy. .
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http://dx.doi.org/10.1158/2159-8290.CD-16-0611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380483PMC
April 2017

Pentraxin-3 is a PI3K signaling target that promotes stem cell-like traits in basal-like breast cancers.

Sci Signal 2017 02 21;10(467). Epub 2017 Feb 21.

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

Basal-like breast cancers (BLBCs) exhibit hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway because of the frequent mutational activation of the catalytic subunit and the genetic loss of its negative regulators PTEN (phosphatase and tensin homolog) and INPP4B (inositol polyphosphate-4-phosphatase type II). However, PI3K inhibitors have had limited clinical efficacy in BLBC management because of compensatory amplification of PI3K downstream signaling loops. Therefore, identification of critical PI3K mediators is paramount to the development of effective BLBC therapeutics. Using transcriptomic analysis of activated PIK3CA-expressing BLBC cells, we identified the gene encoding the humoral pattern recognition molecule pentraxin-3 (PTX3) as a critical target of oncogenic PI3K signaling. We found that PTX3 abundance is stimulated, in part, through AKT- and nuclear factor κB (NF-κB)-dependent pathways and that presence of PTX3 is necessary for PI3K-induced stem cell-like traits. We further showed that expression is greater in tumor samples from patients with BLBC and that it is prognostic of poor patient survival. Our results thus reveal PTX3 as a newly identified PI3K-regulated biomarker and a potential therapeutic target in BLBC.
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http://dx.doi.org/10.1126/scisignal.aah4674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858194PMC
February 2017

The SCFβ-TRCP E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to promote hepatic lipogenesis.

Sci Signal 2017 01 3;10(460). Epub 2017 Jan 3.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

The SCF E3 ubiquitin ligase complex plays pivotal roles in normal cellular physiology and in pathophysiological conditions. Identification of β-transducin repeat-containing protein (β-TRCP) substrates is therefore critical to understand SCF biology and function. We used a β-TRCP-phosphodegron motif-specific antibody in a β-TRCP substrate screen coupled with tandem mass spectrometry and identified multiple β-TRCP substrates. One of these substrates was Lipin1, an enzyme and suppressor of the family of sterol regulatory element-binding protein (SREBP) transcription factors, which activate genes encoding lipogenic factors. We showed that SCF specifically interacted with and promoted the polyubiquitination of Lipin1 in a manner that required phosphorylation of Lipin1 by mechanistic target of rapamycin 1 (mTORC1) and casein kinase I (CKI). β-TRCP depletion in HepG2 hepatocellular carcinoma cells resulted in increased Lipin1 protein abundance, suppression of SREBP-dependent gene expression, and attenuation of triglyceride synthesis. Moreover, β-TRCP1 knockout mice showed increased Lipin1 protein abundance and were protected from hepatic steatosis induced by a high-fat diet. Together, these data reveal a critical physiological function of β-TRCP in regulating hepatic lipid metabolic homeostasis in part through modulating Lipin1 stability.
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http://dx.doi.org/10.1126/scisignal.aah4117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215841PMC
January 2017

Aspirin Suppresses Growth in PI3K-Mutant Breast Cancer by Activating AMPK and Inhibiting mTORC1 Signaling.

Cancer Res 2017 02 9;77(3):790-801. Epub 2016 Dec 9.

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Boston, Massachusetts.

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiologic studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA Here, we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on COX-2 and NF-κB. We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mTORC1 inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, whereas combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports the evaluation of aspirin and PI3K pathway inhibitors as a combination therapy for targeting breast cancer. Cancer Res; 77(3); 790-801. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-2400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290090PMC
February 2017

LINC00520 is induced by Src, STAT3, and PI3K and plays a functional role in breast cancer.

Oncotarget 2016 Dec;7(50):81981-81994

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Long non-coding RNAs (lncRNAs) have been implicated in normal cellular homeostasis as well as pathophysiological conditions, including cancer. Here we performed global gene expression profiling of mammary epithelial cells transformed by oncogenic v-Src, and identified a large subset of uncharacterized lncRNAs potentially involved in breast cancer development. Specifically, our analysis revealed a novel lncRNA, LINC00520 that is upregulated upon ectopic expression of oncogenic v-Src, in a manner that is dependent on the transcription factor STAT3. Similarly, LINC00520 is also increased in mammary epithelial cells transformed by oncogenic PI3K and its expression is decreased upon knockdown of mutant PIK3CA. Additional expression profiling highlight that LINC00520 is elevated in a subset of human breast carcinomas, with preferential enrichment in the basal-like molecular subtype. ShRNA-mediated depletion of LINC00520 results in decreased cell migration and loss of invasive structures in 3D. RNA sequencing analysis uncovers several genes that are differentially expressed upon ectopic expression of LINC00520, a significant subset of which are also induced in v-Src-transformed MCF10A cells. Together, these findings characterize LINC00520 as a lncRNA that is regulated by oncogenic Src, PIK3CA and STAT3, and which may contribute to the molecular etiology of breast cancer.
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http://dx.doi.org/10.18632/oncotarget.11962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347668PMC
December 2016

pVHL suppresses kinase activity of Akt in a proline-hydroxylation-dependent manner.

Science 2016 08;353(6302):929-32

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

Activation of the serine-threonine kinase Akt promotes the survival and proliferation of various cancers. Hypoxia promotes the resistance of tumor cells to specific therapies. We therefore explored a possible link between hypoxia and Akt activity. We found that Akt was prolyl-hydroxylated by the oxygen-dependent hydroxylase EglN1. The von Hippel-Lindau protein (pVHL) bound directly to hydroxylated Akt and inhibited Akt activity. In cells lacking oxygen or functional pVHL, Akt was activated to promote cell survival and tumorigenesis. We also identified cancer-associated Akt mutations that impair Akt hydroxylation and subsequent recognition by pVHL, thus leading to Akt hyperactivation. Our results show that microenvironmental changes, such as hypoxia, can affect tumor behaviors by altering Akt activation, which has a critical role in tumor growth and therapeutic resistance.
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http://dx.doi.org/10.1126/science.aad5755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326551PMC
August 2016

Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

Mol Cell 2016 06 26;62(6):929-942. Epub 2016 May 26.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

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 the 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 or 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.
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http://dx.doi.org/10.1016/j.molcel.2016.04.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912424PMC
June 2016

Glutathione biosynthesis is a metabolic vulnerability in PI(3)K/Akt-driven breast cancer.

Nat Cell Biol 2016 05 18;18(5):572-8. Epub 2016 Apr 18.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

Cancer cells often select for mutations that enhance signalling through pathways that promote anabolic metabolism. Although the PI(3)K/Akt signalling pathway, which is frequently dysregulated in breast cancer, is a well-established regulator of central glucose metabolism and aerobic glycolysis, its regulation of other metabolic processes required for tumour growth is not well defined. Here we report that in mammary epithelial cells, oncogenic PI(3)K/Akt stimulates glutathione (GSH) biosynthesis by stabilizing and activating NRF2 to upregulate the GSH biosynthetic genes. Increased NRF2 stability is dependent on the Akt-mediated accumulation of p21(Cip1/WAF1) and GSK-3β inhibition. Consistently, in human breast tumours, upregulation of NRF2 targets is associated with PI(3)K pathway mutation status and oncogenic Akt activation. Elevated GSH biosynthesis is required for PI(3)K/Akt-driven resistance to oxidative stress, initiation of tumour spheroids, and anchorage-independent growth. Furthermore, inhibition of GSH biosynthesis with buthionine sulfoximine synergizes with cisplatin to selectively induce tumour regression in PI(3)K pathway mutant breast cancer cells, both in vitro and in vivo. Our findings provide insight into GSH biosynthesis as a metabolic vulnerability associated with PI(3)K pathway mutant breast cancers.
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http://dx.doi.org/10.1038/ncb3341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848114PMC
May 2016

Oncogenic AKT1(E17K) mutation induces mammary hyperplasia but prevents HER2-driven tumorigenesis.

Oncotarget 2016 Apr;7(14):17301-13

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

One of the most frequently deregulated signaling pathways in breast cancer is the PI 3-K/Akt cascade. Genetic lesions are commonly found in PIK3CA, PTEN, and AKT, which lead to excessive and constitutive activation of Akt and downstream signaling that results in uncontrolled proliferation and increased cellular survival. One such genetic lesion is the somatic AKT1(E17K) mutation, which has been identified in 4-8% of breast cancer patients. To determine how this mutation contributes to mammary tumorigenesis, we constructed a genetically engineered mouse model that conditionally expresses human AKT1(E17K) in the mammary epithelium. Although AKT1(E17K) is only weakly constitutively active and does not promote proliferation in vitro, it is capable of escaping negative feedback inhibition to exhibit sustained signaling dynamics in vitro. Consistently, both virgin and multiparous AKT1(E17K) mice develop mammary gland hyperplasia that do not progress to carcinoma. This hyperplasia is accompanied by increased estrogen receptor expression, although exposure of the mice to estrogen does not promote tumor development. Moreover, AKT1(E17K) prevents HER2-driven mammary tumor formation, in part through negative feedback inhibition of RTK signaling. Analysis of TCGA breast cancer data revealed that the mRNA expression, total protein levels, and phosphorylation of various RTKs are decreased in human tumors harboring AKT1(E17K).
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http://dx.doi.org/10.18632/oncotarget.8191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4951213PMC
April 2016

Nonessential amino acid metabolism in breast cancer.

Adv Biol Regul 2016 09 21;62:11-17. Epub 2016 Jan 21.

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, USA. Electronic address:

Interest in studying cancer metabolism has risen in recent years, as it has become evident that the relationship between cancer and metabolic pathways could reveal novel biomarkers and therapeutic targets. Metabolic starvation therapy is particularly promising due to its low toxicity. Nonessential amino acids are promising metabolites for such therapy because they become essential in many tumor cells, including breast cancer cells. This review will focus on four nonessential amino acid metabolism pathways: glutamine-glutamate, serine-glycine, cysteine, and arginine-proline metabolism. Recent studies of these amino acids have revealed metabolic enzymes that have the potential to be effective as cancer therapy targets or biomarkers for response to metabolic starvation therapy. The review will also discuss features of nonessential amino acid metabolism that merit further investigation to determine their relevancy to breast cancer treatment.
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http://dx.doi.org/10.1016/j.jbior.2016.01.001DOI Listing
September 2016

PtdIns(3,4,5)P3-Dependent Activation of the mTORC2 Kinase Complex.

Cancer Discov 2015 Nov 20;5(11):1194-209. Epub 2015 Aug 20.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Unlabelled: 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.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631654PMC
November 2015

PIPPing on AKT1: How Many Phosphatases Does It Take to Turn off PI3K?

Cancer Cell 2015 Aug;28(2):143-5

Department of Medicine, Boston University School of Medicine, 650 Albany Street, Boston, MA, 02118, USA. Electronic address:

In this issue of Cancer Cell, Ooms and colleagues show that the lipid phosphatase PIPP/INPP5J, frequently inactivated in triple-negative breast cancers, functions as a tumor suppressor by specifically modulating the activity of AKT1 in the context of oncogenic PI3K signaling, leading to inhibition of metastatic dissemination.
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http://dx.doi.org/10.1016/j.ccell.2015.07.010DOI Listing
August 2015

MERIT40 Is an Akt Substrate that Promotes Resolution of DNA Damage Induced by Chemotherapy.

Cell Rep 2015 Jun 28;11(9):1358-66. Epub 2015 May 28.

Departments of Pathology and Medicine and Cancer Center, Beth Israel Deaconess Medical Center (BIDMC), Harvard Medical School, Boston, MA 02215, USA. Electronic address:

Resistance to cytotoxic chemotherapy drugs, including doxorubicin, is a significant obstacle to the effective treatment of breast cancer. Here, we have identified a mechanism by which the PI3K/Akt pathway mediates resistance to doxorubicin. In addition to inducing DNA damage, doxorubicin triggers sustained activation of Akt signaling in breast cancer cells. We show that Akt contributes to chemotherapy resistance such that PI3K or Akt inhibitors sensitize cells to doxorubicin. We identify MERIT40, a component of the BRCA1-A DNA damage repair complex, as an Akt substrate that is phosphorylated following doxorubicin treatment. MERIT40 phosphorylation facilitates assembly of the BRCA1-A complex in response to DNA damage and contributes to DNA repair and cell survival following doxorubicin treatment. Finally, MERIT40 phosphorylation in human breast cancers is associated with estrogen receptor positivity. Our findings suggest that combination therapy with PI3K or Akt inhibitors and doxorubicin may constitute a successful strategy for overcoming chemotherapy resistance.
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http://dx.doi.org/10.1016/j.celrep.2015.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4464897PMC
June 2015

The phosphoinositide 3-kinase pathway and therapy resistance in cancer.

F1000Prime Rep 2015 3;7:13. Epub 2015 Feb 3.

Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School 330 Brookline Avenue, EC/CLS 633A, Boston, MA 02215 USA.

The phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling network is a master regulator of processes that contribute to tumorigenesis and tumor maintenance. The PI3K pathway also plays a critical role in driving resistance to diverse anti-cancer therapies. This review article focuses on mechanisms by which the PI3K pathway contributes to therapy resistance in cancer, and highlights potential combination therapy strategies to circumvent resistance driven by PI3K signaling. In addition, resistance mechanisms that limit the clinical efficacy of small molecule inhibitors of the PI3K pathway are discussed.
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http://dx.doi.org/10.12703/P7-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335789PMC
March 2015

NFAT1 promotes intratumoral neutrophil infiltration by regulating IL8 expression in breast cancer.

Mol Oncol 2015 Jun 19;9(6):1140-54. Epub 2015 Feb 19.

Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA. Electronic address:

NFAT transcription factors are key regulators of gene expression in immune cells. In addition, NFAT1-induced genes play diverse roles in mediating the progression of various solid tumors. Here we show that NFAT1 induces the expression of the IL8 gene by binding to its promoter and leading to IL8 secretion. Thapsigargin stimulation of breast cancer cells induces IL8 expression in an NFAT-dependent manner. Moreover, we show that NFAT1-mediated IL8 production promotes the migration of primary human neutrophils in vitro and also promotes neutrophil infiltration in tumor xenografts. Furthermore, expression of active NFAT1 effectively suppresses the growth of nascent and established tumors by a non cell-autonomous mechanism. Evaluation of breast tumor tissue reveals that while the levels of NFAT1 are similar in tumor cells and normal breast epithelium, cells in the tumor stroma express higher levels of NFAT1 compared to normal stroma. Elevated levels of NFAT1 also correlate with increased neutrophil infiltrate in breast tumors. These data point to a mechanism by which NFAT1 orchestrates the communication between breast cancer cells and host neutrophils during breast cancer progression.
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http://dx.doi.org/10.1016/j.molonc.2015.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439371PMC
June 2015

SGK3 mediates INPP4B-dependent PI3K signaling in breast cancer.

Mol Cell 2014 Nov 30;56(4):595-607. Epub 2014 Oct 30.

Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

Oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), occur with high frequency in breast cancer. The protein kinase Akt is considered to be the primary effector of PIK3CA, although mechanisms by which PI3K mediates Akt-independent tumorigenic signals remain obscure. We show that serum and glucocorticoid-regulated kinase 3 (SGK3) is amplified in breast cancer and activated downstream of PIK3CA in a manner dependent on the phosphoinositide phosphatase INPP4B. Expression of INPP4B leads to enhanced SGK3 activation and suppression of Akt phosphorylation. Activation of SGK3 downstream of PIK3CA and INPP4B is required for 3D proliferation, invasive migration, and tumorigenesis in vivo. We further show that SGK3 targets the metastasis suppressor NDRG1 for degradation by Fbw7. We propose a model in which breast cancers harboring oncogenic PIK3CA activate SGK3 signaling while suppressing Akt, indicative of oncogenic functions for both INPP4B and SGK3 in these tumors.
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http://dx.doi.org/10.1016/j.molcel.2014.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255362PMC
November 2014