Downregulation of PHLPP expression contributes to hypoxia-induced resistance to

MCB Accepts, published online ahead of print on 23 September 2013 Mol. Cell. Biol. doi:10.1128/MCB.00695-13 Copyright © 2013, American Society for Mic...
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MCB Accepts, published online ahead of print on 23 September 2013 Mol. Cell. Biol. doi:10.1128/MCB.00695-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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Downregulation of PHLPP expression contributes to hypoxia-induced resistance to

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chemotherapy in colon cancer cells

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Yang-An Wen1, Payton D. Stevens1, Michael L. Gasser2, Romina Andrei2, and Tianyan Gao*1,3

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Markey Cancer Center, 2Department of Biology, and 3Department of Molecular and Cellular

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Biochemistry, University of Kentucky, Lexington, KY 40536-0509

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* Corresponding author:

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Tianyan Gao, Department of Molecular and Cellular Biochemistry, University of Kentucky,

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Lexington,

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[email protected]

KY

40536-0509,

Tel:

859-323-3454;

Fax:

859-323-2074;

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Running Title: Downregulation of PHLPP by hypoxia induces chemoresistance

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Word count: 36,808

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Abstract: 194

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Email:

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Abstract

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Hypoxia is a feature of solid tumors. Most tumors are at least partially hypoxic. This hypoxic

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environment plays a critical role in promoting resistance to anticancer drugs. PHLPP, a novel

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family of Ser/Thr protein phosphatases, functions as a tumor suppressor in colon cancers. Here,

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we show that the expression of both PHLPP isoforms is negatively regulated by hypoxia/anoxia

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in colon cancer cells. Interestingly, hypoxia-induced decrease of PHLPP expression is attenuated

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by knocking down HIF1α but not HIF2α. Whereas the mRNA levels of PHLPP are not

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significantly altered by oxygen deprivation, the reduction of PHLPP expression is caused by

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decreased protein translation downstream of mTOR and increased degradation. Specifically,

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hypoxia-induced downregulation of PHLPP is partially rescued in TSC2 or 4E-BP1 knockdown

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cells as the result of elevated mTOR activity and protein synthesis. Moreover, oxygen

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deprivation destabilizes PHLPP protein by decreasing the expression of USP46, a deubiquitinase

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of PHLPP. Functionally, downregulation of PHLPP contributes to hypoxia-induced

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chemoresistance in colon cancer cells. Taken together, we have identified hypoxia as a novel

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mechanism by which PHLPP is downregulated in colon cancer, and the expression of PHLPP

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may serve as a biomarker for better understanding chemoresistance in cancer treatment.

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Introduction

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Hypoxia is a condition commonly occurring in most solid tumors. Numerous studies have

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demonstrated that hypoxia plays a pivotal role in tumor progression and metastasis and is often

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associated with increased malignancy and poor prognosis (24, 33). Serving as the primary

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modulators of hypoxic stress, hypoxia-inducible factors (HIFs) are rapidly induced in response to

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oxygen deprivation to regulate the expression of genes that facilitate adaptation to hypoxic

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conditions (26, 33). The HIF transcription factors are heterodimers consisting of an O2-sensitive

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α subunit and a stable β subunit. There are three isoforms of mammalian HIFα, of which HIF1α

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and HIF2α are the most structurally similar and best characterized (26). Under normoxic

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condition, HIFs are modified at two conserved proline residues by prolyl hydroxylase domain

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proteins (PHDs, consisting of PHD1, PHD2 and PHD3). The modified HIFs are subsequently

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recognized and ubiquitinated by an E3 ligase, the von Hippel-Lindau (VHL) protein, resulting in

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the degradation of HIF proteins via the proteasome pathway. Under hypoxic conditions, the

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hydroxylase activity of PHDs is inhibited and stabilized HIF transcription factors can translocate

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to the nucleus and regulate the transcription of hypoxia-associated genes (14, 26, 28).

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Previous studies have shown that hypoxia inhibits mTOR and cap-dependent protein

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translation via a TSC2-dependent mechanism upon the induction of REDD1 (5, 39). mTOR is an

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evolutionarily conserved a serine/threonine protein kinase that functions in two distinct

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complexes, mTORC1 and mTORC2, in cells. mTORC1 serves as a nutrient and energy sensor

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by controlling protein translation through phosphorylation of 4E-BP1 and S6K1 (25). The 3

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inhibition of mTORC1-dependent protein translation has been suggested to affect cellular

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tolerance to hypoxia; however, it remains elusive how hypoxia-induced downregulation of

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translation contributes to tumor progression. Furthermore, cancer cells exposed to hypoxic

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conditions are notoriously known to be more resistant to apoptosis induced by radiation and

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chemotherapy drugs, and a variety of mechanisms have been suggested to affect the treatment

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sensitivity (4, 10, 20, 38). Thus, a better understanding of hypoxia-mediated chemoresistance is

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needed in order to treat cancer cells more effectively.

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PHLPP (PH domain Leucine-rich-repeats Protein Phosphatase) belongs to a novel family of

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Ser/Thr protein phosphatases consisting of two isoforms namely PHLPP1 and PHLPP2. As a

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direct regulator of several critical protein kinases including Akt, protein kinase C (PKC), MAPK,

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and Mst1, PHLPP plays a critical role in maintaining the balance of signaling and homeostasis in

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cells (2, 32, 34, 37). Recently, an increasing numbers of studies have revealed that loss of

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PHLPP expression is highly associated with tumor progression both in human cancers and in

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mouse models, thus further confirming the role of PHLPP as a tumor suppressor (7, 22, 29, 30).

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Our previous studies have shown that PHLPP is degraded via the ubiquitin-proteasome pathway,

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and the turnover of PHLPP proteins is mediated by an E3 ubiquitin ligase β-TrCP (17).

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Conversely, the expression of PHLPP is stabilized by a deubiquitinase USP46, which rescues

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PHLPP from proteasomal degradation by removing ubiquitin chains from PHLPP directly (19).

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In this study, we investigated the role of hypoxia in negatively controlling the expression of

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PHLPP in colon cancer cells. Our results revealed the functional importance of hypoxia-

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mediated downregulation of PHLPP in regulating chemosensitivity.

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Materials and Methods

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Antibodies and reagents

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Antibodies against PHLPP1 and PHLPP2 were purchased from Bethyl Laboratory. The HIF1α

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antibody was obtained from BD Biosciences, and antibodies against HIF-2α and ubiquitin were

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from Santa Cruz Biotechnology. The carbonic anhydrase IX (CA9) antibody was from Novus

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Biologicals; the β-TrCP antibody was from Invitrogen; and the USP46 and -tubulin antibodies

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were from Sigma-Aldrich. The following antibodies against 4E-BP1, TSC2, phospho-S6K

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(T389), S6K1, and PHD2 were from Cell Signaling. The anti-HA Affinity Matrix of rat IgG1

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was purchased from Roche Applied Science. The expression plasmids for HA-tagged PHLPP1

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and PHLPP2 have been described in previous studies (3, 12, 22). The Myc-tagged USP46 was

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constructed by subcloning of USP46 coding sequence into the pcDNA4-Myc/His vector.

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Cell culture and treatment

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Human colon cancer cell lines SW480 and HCT116 were cultured in Dulbecco modified Eagle

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medium (DMEM, Cellgro) and McCoy’s 5A, respectively. All media were supplemented with

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10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin. Stable SW480

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cells overexpressing HA-PHLPP1 or HA-PHLPP2 were generated as described previously (21). 5

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Cells were routinely cultured in a humidified incubator at 37 ºC in 95% air–5% CO2. The

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hypoxic condition was achieved by placing cells in a sealed hypoxia incubator chamber

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(Stemcell technologies Inc.) filled with 5% CO2 and 95% N2. Alternatively, cells were grown in

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a humidified incubator maintained with 5% CO2 and 1% O2. To induce hypoxia chemically, cells

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were treated with desferrioxamine (DFO) or CoCl2 for the indicated times.

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Lentivirus-mediated delivery of shRNA

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The stable PHLPP1, PHLPP2, 4E-BP1, and TSC2 knockdown HCT116 and SW480 cells were

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generated in previous studies using lentivirus carrying specific shRNA targeting sequences for

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each genes (16, 21, 22). To generate stable knockdown cells for HIF1α, HIF2α, and PHD2, the

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shRNA for each gene was constructed in pLKO.1-puro vector and purchased from Sigma-

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Aldrich. The shRNA targeting sequences are as the following:

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CCAGTTATGATTGTGAAGTTA-3’ (#1), and 5’-CGGCGAAGTAAAGAATCTGAA-3’ (#2);

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for

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GCGCAAATGTACCCAATGATA-3’

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ACGCCACTGTAACGGGAAGCT-3’ (#1), and 5’-TGCACGACACCGGGAAGTTCA-3’ (#2).

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The lentivirus-mediated delivery of shRNA and selection for stable knockdown cells were

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carried out as previously described (22).

HIF2α,

5’-CGACCTGAAGATTGAAGTGAT-3’ (#2);

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Immunoblotting 6

and

(#1), for

for HIF1α, 5’-

and

5’-

PHD2,

5’-

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Cultured cells were harvested and lysed in Lysis Buffer (50 mM Na2HPO4, 1 mM sodium

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pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 1 mM DTT, 200

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mM benzamidine, 40 mg ml-1 leupeptin, 200 mM PMSF) and the detergent-solublized cell

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lysates were obtained after centrifugation for 5 minutes at 16,000 g at 4 oC. Equal amounts of

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cell lysates as determined by Bradford assays were resolved by SDS-PAGE and subjected to

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immunoblotting analysis. The density of ECL signals was obtained and quantified using a

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FluoChem digital imaging system (Alpha Innotech).

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Analysis of ubiquitination

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To examine the ubiquitination of PHLPP in cells, stable SW480 cells expressing vector, HA-

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PHLPP1, or HA-PHLPP2 cultured cells were exposed to normoxia or hypoxia for 6 hours and

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lysed in Buffer A (50 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 1 mM

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DTT, 200 µM benzamidine, 40 µg ml-1 leupeptin, 200 µM PMSF, and 10 mM N-

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Ethylmaleimide). The detergent-solubilized cell lysates were incubated with the anti-HA Affinity

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Matrix at 4 oC for overnight. The beads were washed three times with Buffer B (Buffer A plus

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250 mM NaCl) and once with Buffer A. The immunoprecipitated proteins were analyzed by

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SDS-PAGE and immunoblotting using ubiquitin and HA antibodies.

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Cell viability assay

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Equal numbers of HCT116 cells (2,000 cells / well) were seeded onto 96-well plates and allowed

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to attach overnight. Cells were then treated with different concentrations of paclitaxel or

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oxaliplatin and cultured under normoxic or hypoxic conditions for additional 48 hours. MTS

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assays were performed according to the manufacturer’s protocol (Promega). The absorbance was

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measured at OD492 using a microplate reader.

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Real-time PCR

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Total RNA was isolated with RNeasy kit (Qiagen) from SW480 and HCT116 cells following

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specific treatment. Equal amounts of RNA were used as templates for the synthesis of cDNA

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using High Capacity cDNA Reverse Transcription kit (Applied Biosysems). Real-time PCR

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reaction was performed using PHLPP1-, PHLPP2-, or USP46-specific probes using StepOne

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Real-Time PCR system (Applied Biosysems). All values were normalized to the level of β-actin.

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Results

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Expression of PHLPP is downregulated under hypoxic conditions in colon cancer cells

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Since hypoxia and expression of HIFs have been linked to the development and progression

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of colon cancer, we first investigated the effect of oxygen deprivation on the expression of

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PHLPP isoforms in colon cancer cells. As 50–60% of solid tumors are estimated to contain areas

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of hypoxic and/or anoxic tissues (36), we performed the experiments under both 1% O2 and 8

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anoxic condition (