bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2020‒11‒08
twenty-two papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research


  1. Mol Cell. 2020 Oct 26. pii: S1097-2765(20)30693-6. [Epub ahead of print]
    Takahashi N, Cho P, Selfors LM, Kuiken HJ, Kaul R, Fujiwara T, Harris IS, Zhang T, Gygi SP, Brugge JS.
      Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.
    Keywords:  oxidative stress, cancer, cell death, ferroptosis, 3D culture, NRF2, CRISPR screening, selenoprotein, GPX4
    DOI:  https://doi.org/10.1016/j.molcel.2020.10.010
  2. Cell Metab. 2020 Nov 03. pii: S1550-4131(20)30538-6. [Epub ahead of print]32(5): 889-900.e7
    Ludikhuize MC, Meerlo M, Gallego MP, Xanthakis D, Burgaya Julià M, Nguyen NTB, Brombacher EC, Liv N, Maurice MM, Paik JH, Burgering BMT, Rodriguez Colman MJ.
      Differential WNT and Notch signaling regulates differentiation of Lgr5+ crypt-based columnar cells (CBCs) into intestinal cell lineages. Recently we showed that mitochondrial activity supports CBCs, while adjacent Paneth cells (PCs) show reduced mitochondrial activity. This implies that CBC differentiation into PCs involves a metabolic transition toward downregulation of mitochondrial dependency. Here we show that Forkhead box O (FoxO) transcription factors and Notch signaling interact in determining CBC fate. In agreement with the organoid data, Foxo1/3/4 deletion in mouse intestine induces secretory cell differentiation. Importantly, we show that FOXO and Notch signaling converge on regulation of mitochondrial fission, which in turn provokes stem cell differentiation into goblet cells and PCs. Finally, scRNA-seq-based reconstruction of CBC differentiation trajectories supports the role of FOXO, Notch, and mitochondria in secretory differentiation. Together, this points at a new signaling-metabolic axis in CBC differentiation and highlights the importance of mitochondria in determining stem cell fate.
    Keywords:  FOXO; Notch; differentiation; intestine; metabolism; mitochondria; stem cells
    DOI:  https://doi.org/10.1016/j.cmet.2020.10.005
  3. Cell Metab. 2020 Nov 03. pii: S1550-4131(20)30549-0. [Epub ahead of print]32(5): 699-701
    Xu K, Shyu A, Li MO.
      Nutrient acquisition and metabolism are integral components of cell growth, proliferation, and differentiation programs. In a recent study in Nature, Bian et al. (2020) revealed that cancer cells outcompete T cells for methionine uptake, resulting in diminished SAM production, attenuated H3K79 dimethylation, decreased STAT5 expression, and impaired T cell immunity to cancer.
    DOI:  https://doi.org/10.1016/j.cmet.2020.10.016
  4. Cancer Discov. 2020 Nov 02.
    Fendt SM, Frezza C, Erez A.
      Cancer cells continuously rewire their metabolism to fulfill their need for rapid growth and survival while subject to changes in environmental cues. Thus, a vital component of a cancer cell lies in its metabolic adaptability. The constant demand for metabolic alterations requires flexibility, that is, the ability to utilize different metabolic substrates; as well as plasticity, that is, the ability to process metabolic substrates in different ways. In this review, we discuss how dynamic changes in cancer metabolism affect tumor progression and the consequential implications for cancer therapy. SIGNIFICANCE: Recognizing cancer dynamic metabolic adaptability as an entity can lead to targeted therapy that is expected to decrease drug resistance.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-0844
  5. J Cell Mol Med. 2020 Nov 03.
    Xu L, Chen J, Jia L, Chen X, Awaleh Moumin F, Cai J.
      Gastric cancer is a major cause of mortality worldwide. The glutamate/aspartate transporter SLC1A3 has been implicated in tumour metabolism and progression, but the roles of SLC1A3 in gastric cancer remain unclear. We used bioinformatics approaches to analyse the expression of SLC1A3 and its role in gastric cancer. The expression levels of SLC1A3 were examined using RT-qPCR and Western bolting. SLC1A3 overexpressing and knock-down cell lines were constructed, and the cell viability was evaluated. Glucose consumption, lactate excretion and ATP levels were determined. The roles of SLC1A3 in tumour growth were evaluated using a xenograft tumour growth model. SLC1A3 was found to be overexpressed in gastric cancer, and this overexpression was associated with poor prognosis. In vitro and in vivo assays showed that SLC1A3 affected glucose metabolism and promoted gastric cancer growth. GSEA analysis suggested that SLC1A3 was positively associated with the up-regulation of the PI3K/AKT pathway. SLC1A3 overexpression activated the PI3K/AKT pathway and up-regulated GLUT1, HK II and LDHA expression. The PI3K/AKT inhibitor LY294002 prevented SLC1A3-induced glucose metabolism and cell proliferation. Our findings indicate that SLC1A3 promotes gastric cancer progression via the PI3K/AKT signalling pathway. SLC1A3 is therefore a potential therapeutic target in gastric cancer.
    Keywords:  PI3K/AKT; SLC1A3; bioinformatics; gastric cancer; glucose metabolism; prognosis
    DOI:  https://doi.org/10.1111/jcmm.16060
  6. Cell. 2020 Oct 28. pii: S0092-8674(20)31322-2. [Epub ahead of print]
    Banh RS, Biancur DE, Yamamoto K, Sohn ASW, Walters B, Kuljanin M, Gikandi A, Wang H, Mancias JD, Schneider RJ, Pacold ME, Kimmelman AC.
      Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
    Keywords:  mRNA translation; metabolic crosstalk; neurons; pancreatic cancer; serine
    DOI:  https://doi.org/10.1016/j.cell.2020.10.016
  7. Cancers (Basel). 2020 Nov 04. pii: E3249. [Epub ahead of print]12(11):
    Zhang Y, Wu ZX, Yang Y, Wang JQ, Li J, Sun Z, Teng QX, Ashby CR, Yang DH.
      Colorectal cancer (CRC) is a leading cause of cancer deaths in the United States. Currently, chemotherapy is a first-line treatment for CRC. However, one major drawback of chemotherapy is the emergence of multidrug resistance (MDR). It has been well-established that the overexpression of the ABCB1 and/or ABCG2 transporters can produce MDR in cancer cells. In this study, we report that in vitro, poziotinib can antagonize both ABCB1- and ABCG2-mediated MDR at 0.1-0.6 μM in the human colon cancer cell lines, SW620/Ad300 and S1-M1-80. Mechanistic studies indicated that poziotinib increases the intracellular accumulation of the ABCB1 transporter substrates, paclitaxel and doxorubicin, and the ABCG2 transporter substrates, mitoxantrone and SN-38, by inhibiting their substrate efflux function. Accumulation assay results suggested that poziotinib binds reversibly to the ABCG2 and ABCB1 transporter. Furthermore, western blot experiments indicated that poziotinib, at 0.6 μM, significantly downregulates the expression of the ABCG2 but not the ABCB1 transporter protein, suggesting that the ABCG2 reversal effect produced by poziotinib is due to transporter downregulation and inhibition of substrate efflux. Poziotinib concentration-dependently stimulated the ATPase activity of both ABCB1 and ABCG2, with EC50 values of 0.02 μM and 0.21 μM, respectively, suggesting that it interacts with the drug-substrate binding site. Molecular docking analysis indicated that poziotinib binds to the ABCB1 (-6.6 kcal/mol) and ABCG2 (-10.1 kcal/mol) drug-substrate binding site. In summary, our novel results show that poziotinib interacts with the ABCB1 and ABCG2 transporter, suggesting that poziotinib may increase the efficacy of certain chemotherapeutic drugs used in treating MDR CRC.
    Keywords:  ABCB1; ABCG2; EGFR inhibitor; colorectal cancer; multidrug resistance; poziotinib
    DOI:  https://doi.org/10.3390/cancers12113249
  8. Clin Cancer Res. 2020 Nov 04. pii: clincanres.2657.2020. [Epub ahead of print]
    Jhaveri K, Chang MT, Juric D, Saura C, Gambardella V, Melnyk A, Patel MR, Ribrag V, Ma CX, Aljumaily R, Bedard PL, Sachdev JC, Dunn L, Won HH, Bond J, Jones S, Savage HM, Scaltriti M, Wilson TR, Wei MC, Hyman DM.
      PURPOSE: Somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), are found in multiple human cancers. While recurrent mutations in PIK3CA helical, regulatory, and kinase domains lead to constitutive PI3K pathway activation, other mutations remain uncharacterized. To further evaluate their clinical actionability, we designed a basket study for patients with PIK3CA-mutant cancers with the isoform-specific PI3K inhibitor, taselisib.PATIENTS AND METHODS: Patients were enrolled based on local PIK3CA mutation testing into 1 of 11 histology-specific cohorts and treated with taselisib at 6 mg or 4 mg daily until progression. Tumor DNA from baseline and progression (when available) was sequenced using a next-generation sequencing panel. Exploratory analyses correlating genomic alterations with treatment outcomes were performed.
    RESULTS: 166 patients with PIK3CA-mutant cancers were enrolled. The confirmed response rate was 9.0%. Activity varied by tumor type and mutant allele, with confirmed responses observed in head and neck squamous (15.4%), cervical (10.0%), and other cancers, plus tumors containing helical domain mutations. Genomic analyses identified mutations potentially associated with resistance to PI3K inhibition upfront (TP53 and PTEN) and post-progression through reactivation of the PI3K pathway (PTEN, STK11, and PIK3R1). Higher rates of dose modification occurred at higher doses of taselisib, indicating a narrow therapeutic index.
    CONCLUSIONS: Taselisib had limited activity in the tumor types tested and is no longer in development. This genome-driven study improves understanding of the activity, limitations, and resistance mechanisms of using PI3K inhibitors as monotherapy to target PIK3CA-mutant tumors.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-20-2657
  9. Breast Cancer Res. 2020 11 02. 22(1): 120
    Garrido-Castro AC, Saura C, Barroso-Sousa R, Guo H, Ciruelos E, Bermejo B, Gavilá J, Serra V, Prat A, Paré L, Céliz P, Villagrasa P, Li Y, Savoie J, Xu Z, Arteaga CL, Krop IE, Solit DB, Mills GB, Cantley LC, Winer EP, Lin NU, Rodon J.
      BACKGROUND: Treatment options for triple-negative breast cancer remain limited. Activation of the PI3K pathway via loss of PTEN and/or INPP4B is common. Buparlisib is an orally bioavailable, pan-class I PI3K inhibitor. We evaluated the safety and efficacy of buparlisib in patients with metastatic triple-negative breast cancer.METHODS: This was a single-arm phase 2 study enrolling patients with triple-negative metastatic breast cancer. Patients were treated with buparlisib at a starting dose of 100 mg daily. The primary endpoint was clinical benefit, defined as confirmed complete response (CR), partial response (PR), or stable disease (SD) for ≥ 4 months, per RECIST 1.1. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity. A subset of patients underwent pre- and on-treatment tumor tissue biopsies for correlative studies.
    RESULTS: Fifty patients were enrolled. Median number of cycles was 2 (range 1-10). The clinical benefit rate was 12% (6 patients, all SD ≥ 4 months). Median PFS was 1.8 months (95% confidence interval [CI] 1.6-2.3). Median OS was 11.2 months (95% CI 6.2-25). The most frequent adverse events were fatigue (58% all grades, 8% grade 3), nausea (34% all grades, none grade 3), hyperglycemia (34% all grades, 4% grade 3), and anorexia (30% all grades, 2% grade 3). Eighteen percent of patients experienced depression (12% grade 1, 6% grade 2) and anxiety (10% grade 1, 8% grade 2). Alterations in PIK3CA/AKT1/PTEN were present in 6/27 patients with available targeted DNA sequencing (MSK-IMPACT), 3 of whom achieved SD as best overall response though none with clinical benefit ≥ 4 months. Of five patients with paired baseline and on-treatment biopsies, reverse phase protein arrays (RPPA) analysis demonstrated reduction of S6 phosphorylation in 2 of 3 patients who achieved SD, and in none of the patients with progressive disease.
    CONCLUSIONS: Buparlisib was associated with prolonged SD in a very small subset of patients with triple-negative breast cancer; however, no confirmed objective responses were observed. Downmodulation of key nodes in the PI3K pathway was observed in patients who achieved SD. PI3K pathway inhibition alone may be insufficient as a therapeutic strategy for triple-negative breast cancer.
    TRIAL REGISTRATION: NCT01790932 . Registered on 13 February 2013; NCT01629615 . Registered on 27 June 2012.
    Keywords:  BKM120; Buparlisib; PI3K pathway; Phase 1; Triple-negative breast cancer
    DOI:  https://doi.org/10.1186/s13058-020-01354-y
  10. Proc Natl Acad Sci U S A. 2020 Nov 02. pii: 202017830. [Epub ahead of print]
    Polytarchou C, Hatziapostolou M, Yau TO, Christodoulou N, Hinds PW, Kottakis F, Sanidas I, Tsichlis PN.
      Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant N-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in p53 -/- and INK4a -/-/Arf -/- mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 in p47 phox-/- MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, Akt3 alterations correlate with a higher frequency of mutation of p53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.
    Keywords:  Akt isoforms; DNA damage; NADPH oxidase; cancer; oxidative stress
    DOI:  https://doi.org/10.1073/pnas.2017830117
  11. Cancer Discov. 2020 Nov;10(11): 1632-1634
    Davidsen K, Sullivan LB.
      The chemotherapeutic enzyme asparaginase depletes systemic asparagine to kill cancers; however, its efficacy thus far is limited to a subset of leukemias. Hinze and colleagues identify that inhibiting proteasomal release of asparagine can sensitize colorectal cancers to asparagine depletion, providing a potential avenue to repurpose asparaginase for treatment of solid tumors.See related article by Hinze et al., p. 1690.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-1251
  12. Sci Adv. 2020 Nov;pii: eabc1251. [Epub ahead of print]6(45):
    Scaiola A, Mangia F, Imseng S, Boehringer D, Berneiser K, Shimobayashi M, Stuttfeld E, Hall MN, Ban N, Maier T.
      The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes, and neurological disorders. mTOR exerts its functions in two distinct multiprotein complexes, mTORC1 and mTORC2. Here, we report a 3.2-Å resolution cryo-EM reconstruction of mTORC2. It reveals entangled folds of the defining Rictor and the substrate-binding SIN1 subunits, identifies the carboxyl-terminal domain of Rictor as the source of the rapamycin insensitivity of mTORC2, and resolves mechanisms for mTORC2 regulation by complex destabilization. Two previously uncharacterized small-molecule binding sites are visualized, an inositol hexakisphosphate (InsP6) pocket in mTOR and an mTORC2-specific nucleotide binding site in Rictor, which also forms a zinc finger. Structural and biochemical analyses suggest that InsP6 and nucleotide binding do not control mTORC2 activity directly but rather have roles in folding or ternary interactions. These insights provide a firm basis for studying mTORC2 signaling and for developing mTORC2-specific inhibitors.
    DOI:  https://doi.org/10.1126/sciadv.abc1251
  13. J Photochem Photobiol B. 2020 Oct 15. pii: S1011-1344(20)30505-4. [Epub ahead of print]213 112055
    Ahmed AR, Candeo A, D'Abrantes S, Needham SR, Yadav RB, Botchway SW, Parker AW.
      The range of cellular functions the mechanistic target of rapamycin (mTOR) protein performs makes it an attractive drug target for cancer therapy. However, the cellular localisation and mode of action of second generation inhibitors of mTOR is poorly understood despite the level of attention there is in targeting the mTOR protein. We have therefore studied the properties of the pan-mTOR inhibitor AZD2014, an ideal candidate to study because it is naturally fluorescent, characterising its photochemical properties in solution phase (DMSO, PBS and BSA) and within living cells, where it localises within both the nucleus and the cytoplasm but with different excited state lifetimes of 4.8 (+/- 0.5) and 3.9 (+/- 0.4) ns respectively. We measure the uptake of the inhibitor AZD2014 (7 μM) in monolayer HEK293 cells occurring with a half-life of 1 min but observe complex behaviour for 3D spheroids with the core of the spheroid showing a slower uptake and a slow biphasic behaviour at longer times. From a cellular perspective using fluorescence lifetime imaging microscopy AZD2014 was found to interact directly with GFP-tagged mTORC1 proteins including the downstream target, S6K1. We observe light sensitive behaviour of the cells containing AZD2014 which leads to cell death, in both monolayer and spheroids cells, demonstrating the potential of AZD2014 to act as a possible photodynamic drug under both single photon and multiphoton excitation and discuss its use as a photosensitizer. We also briefly characterise another pan-mTOR inhibitor, INK128.
    Keywords:  AZD2014; FLIM;light sheet fluorescence microscopy; Fluorescence microscopy; GFP; INK128, mTOR; Multiphoton; PDT; Photosensitizer
    DOI:  https://doi.org/10.1016/j.jphotobiol.2020.112055
  14. EMBO Rep. 2020 Nov 07. e49756
    Iacono A, Pompa A, De Marchis F, Panfili E, Greco FA, Coletti A, Orabona C, Volpi C, Belladonna ML, Mondanelli G, Albini E, Vacca C, Gargaro M, Fallarino F, Bianchi R, De Marcos Lousa C, Mazza EM, Bicciato S, Proietti E, Milano F, Martelli MP, Iamandii IM, Graupera Garcia-Mila M, Llena Sopena J, Hawkins P, Suire S, Okkenhaug K, Stark AK, Grassi F, Bellucci M, Puccetti P, Santambrogio L, Macchiarulo A, Grohmann U, Pallotta MT.
      Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.
    Keywords:  dendritic cells; early endosomes; indoleamine 2,3-dioxygenase 1 (IDO1); phosphoinositide 3-kinase (PI3K); tryptophan metabolism
    DOI:  https://doi.org/10.15252/embr.201949756
  15. Nat Commun. 2020 11 03. 11(1): 5559
    van den Boomen DJH, Sienkiewicz A, Berlin I, Jongsma MLM, van Elsland DM, Luzio JP, Neefjes JJC, Lehner PJ.
      Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.
    DOI:  https://doi.org/10.1038/s41467-020-19032-0
  16. Sci Adv. 2020 Nov;pii: eabb7272. [Epub ahead of print]6(45):
    Kong H, Reczek CR, McElroy GS, Steinert EM, Wang T, Sabatini DM, Chandel NS.
      Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness.
    DOI:  https://doi.org/10.1126/sciadv.abb7272
  17. Mol Oncol. 2020 Nov 05.
    Deng R, Guo Y, Li L, He J, Qiang Z, Zhang H, Chen R, Wang Y, Zhao X, Yu J.
      Deubiquitinase BAP1 is an important tumor suppressor in several malignancies, but its functions and critical substrates in prostate cancer (PCa) remain unclear. Here, we report that the mRNA and protein expression levels of BAP1 are downregulated in clinical PCa specimens. BAP1 can physically bind to and deubiquitinate PTEN, which inhibits the ubiquitination-mediated degradation of PTEN and thus stabilizes PTEN protein. Ectopically expressed BAP1 in PCa cells increases PTEN protein level and subsequently inhibits the AKT signaling pathway, thus suppressing PCa progression. Conversely, knockdown of BAP1 in PCa cells leads to the decrease of PTEN protein level and the activation of the Akt signaling pathway, therefore promoting malignant transformation and cancer metastasis. However, these can be reversed by the re-expression of PTEN. More importantly, we found that BAP1 protein level positively correlates with PTEN in a substantial fraction of human cancers. These findings demonstrate that BAP1 is an important deubiquitinase of PTEN for its stability and the BAP1-PTEN signaling axis plays a crucial role in tumor suppression.
    Keywords:  BAP1; PTEN; cancer progression; deubiquitination; prostate cancer
    DOI:  https://doi.org/10.1002/1878-0261.12844
  18. Biochem J. 2020 Nov 04. pii: BCJ20200525. [Epub ahead of print]
    Martín Guerrero SM, Casado P, Hijazi M, Rajeeve V, Plaza J, Abadía-Molina F, Navascués J, Cuadros MA, Cutillas PR, Martín-Oliva D.
      Poly(ADP-ribose) Polymerase 1 (PARP-1) is a nuclear enzyme that catalyse the transfer of ADP-ribose units from NAD+ to several target proteins involved in cellular stress responses. Using WRL68 (HeLa derivate) cells, we previously showed that PARP-1 activation induced by oxidative stress after H2O2 treatment lead to depletion of cellular NAD+ and ATP, which promoted cell death. In this work, LC-MS/MS based phosphoproteomics in WRL68 cells showed that the oxidative damage induced by H2O2 increased the phosphorylation of YAP1, a transcriptional coactivator involved in cell survival, and modified the phosphorylation of other proteins involved in transcription. Genetic or pharmacological inhibition of PARP-1 in H2O2-treated cells reduced YAP1 phosphorylation and degradation and increased cell viability. YAP1 silencing abrogated the protective effect of PARP-1 inhibition, indicating that YAP1 is important for the survival of WRL68 cells exposed to oxidative damage. Supplementation of NAD+ also reduced YAP1 phosphorylation, suggesting that the loss of cellular NAD+ caused by PARP-1 activation after oxidative treatment is responsible for the phosphorylation of YAP1. Finally, PARP-1 silencing after oxidative treatment diminished the activation of the metabolic sensor AMPK. Since, NAD+ supplementation reduced the phosphorylation of some AMPK substrates, we hypothesized that the loss of cellular NAD+ after PARP-1 activation may induce an energy stress that activates AMPK. In summary, we showed a new crucial role of PARP-1 in the response to oxidative stress in which PARP-1 activation reduced cell viability by promoting the phosphorylation and degradation of YAP1 through a mechanism that involves the depletion of NAD+.
    Keywords:  LC-MS/MS; PARP-1; YAP1; cell survival; energy stress; phosphorylation
    DOI:  https://doi.org/10.1042/BCJ20200525
  19. Cancers (Basel). 2020 Oct 29. pii: E3178. [Epub ahead of print]12(11):
    Sinha A, Saleh A, Endersby R, Yuan SH, Chokshi CR, Brown KR, Kuzio B, Kauppinen T, Singh SK, Baker SJ, McKinnon PJ, Katyal S.
      PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors.METHODS: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs.
    RESULTS: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status.
    CONCLUSIONS: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.
    Keywords:  DNA damage; HRD; PARP inhibitor; PTEN; RAD51; RAD51 foci; RNA expression; alkaline comet assay; base excision repair; brain tumor initiating cells; combination therapy; glioblastoma multiforme; homologous recombination; olaparib; synthetic lethality; γH2AX foci
    DOI:  https://doi.org/10.3390/cancers12113178
  20. Cancer Cell. 2020 Oct 26. pii: S1535-6108(20)30539-0. [Epub ahead of print]
    Zhao W, Li J, Chen MM, Luo Y, Ju Z, Nesser NK, Johnson-Camacho K, Boniface CT, Lawrence Y, Pande NT, Davies MA, Herlyn M, Muranen T, Zervantonakis IK, von Euw E, Schultz A, Kumar SV, Korkut A, Spellman PT, Akbani R, Slamon DJ, Gray JW, Brugge JS, Lu Y, Mills GB, Liang H.
      Perturbation biology is a powerful approach to modeling quantitative cellular behaviors and understanding detailed disease mechanisms. However, large-scale protein response resources of cancer cell lines to perturbations are not available, resulting in a critical knowledge gap. Here we generated and compiled perturbed expression profiles of ∼210 clinically relevant proteins in >12,000 cancer cell line samples in response to ∼170 drug compounds using reverse-phase protein arrays. We show that integrating perturbed protein response signals provides mechanistic insights into drug resistance, increases the predictive power for drug sensitivity, and helps identify effective drug combinations. We build a systematic map of "protein-drug" connectivity and develop a user-friendly data portal for community use. Our study provides a rich resource to investigate the behaviors of cancer cells and the dependencies of treatment responses, thereby enabling a broad range of biomedical applications.
    Keywords:  biomarker; cancer signaling pathway; drug response; protein array
    DOI:  https://doi.org/10.1016/j.ccell.2020.10.008
  21. Cancer Sci. 2020 Nov 02.
    Yokogawa T, Yano W, Tsukioka S, Osada A, Wakasa T, Ueno H, Hoshino T, Yamamura K, Fujioka A, Fukuoka M, Ohkubo S, Matsuo K.
      Deficiency in DNA repair proteins confers susceptibility to DNA damage, making cancer cells vulnerable to various cancer chemotherapies. 5-Fluorouracil (5-FU) is an anticancer nucleoside analog that both inhibits thymidylate synthase (TS) and causes DNA damage via the misincorporation of FdUTP and dUTP into DNA under the conditions of dTTP depletion. However, the role of the DNA damage response to its antitumor activity is still unclear. To determine which DNA repair pathway contributes to DNA damage caused by 5-FU and uracil misincorporation, we examined cancer cells treated with 2'-deoxy-5-fluorouridine (FdUrd) in the presence of TAS-114, a highly potent inhibitor of dUTPase that restricts aberrant base misincorporation. Addition of TAS-114 increased FdUTP and dUTP levels in HeLa cells and facilitated 5-FU and uracil misincorporation into DNA, but did not alter TS inhibition or 5-FU incorporation into RNA. TAS-114 showed synergistic potentiation of FdUrd cytotoxicity and caused aberrant base misincorporation, leading to DNA damage and induced cell death even after short-term exposure to FdUrd. Base excision repair (BER) and homologous recombination (HR) were found to be involved in the DNA repair of 5-FU and uracil misincorporation caused by dUTPase inhibition in genetically modified chicken DT40 cell lines and siRNA-treated HeLa cells. These results suggest that BER and HR are major pathways that protect cells from the antitumor effects of massive incorporation of 5-FU and uracil. Further, dUTPase inhibition has the potential to maximize the antitumor activity of fluoropyrimidines in cancers that are defective in BER or HR.
    Keywords:  5-fluorouracil/uracil misincorporation; DNA repair; base excision repair; dUTPase; homologous recombination
    DOI:  https://doi.org/10.1111/cas.14718