bims-pimaco 2021-05-16 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–05–16
24 papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Selective and noncovalent targeting of RAS mutants for inhibition and degradation.
A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background.
The Warburg Effect: Historical Dogma Versus Current Rationale.
Probing RAS Function with Monobodies.
Generation of Patient-Derived Colorectal Cancer Organoids for RAS Studies.
The association between ERK inhibitor sensitivity and molecular characteristics in colorectal cancer.
Mathematical Modeling to Study KRAS Mutant-Specific Responses to Pathway Inhibition.
MiRNA-202-5p promotes Colorectal Carcinogenesis through suppression of PTEN.
Specific activation of glycolytic enzyme enolase 2 in BRAF V600E-mutated colorectal cancer.
Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition.
Folliculin: A Regulator of Transcription Through AMPK and mTOR Signaling Pathways.
Advances and Insights of APC-Asef Inhibitors for Metastatic Colorectal Cancer Therapy.
ArfGAP1 inhibits mTORC1 lysosomal localization and activation.
The chemistry and biology of phosphatidylinositol 4-phosphate at the plasma membrane.
Validation of Isoform- and Mutation-Specific RAS Antibodies.
OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring.
Phosphatidylinositol 3-kinase (PI3K) inhibitors: a recent update on inhibitor design and clinical trials (2016-2020).
A synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3'-phosphatase (PNKP) for targeted therapy of PTEN-deficient colorectal cancer.
The role of GSK3 in metabolic pathway perturbations in cancer.
Cell-cell and cell-substratum contacts in the regulation of MAPK and Akt signalling: Importance in therapy, biopharmacy and bioproduction.
Using BioID to Characterize the RAS Interactome.
DDX54 Plays a Cancerous Role Through Activating P65 and AKT Signaling Pathway in Colorectal Cancer.
Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry.
SLFN5 regulates LAT1-mediated mTOR activation in castration-resistant prostate cancer.

Nat Commun. 2021 May 11. 12(1): 2656

Selective and noncovalent targeting of RAS mutants for inhibition and degradation.

Kai Wen Teng, Steven T Tsai, Takamitsu Hattori, Carmine Fedele, Akiko Koide, Chao Yang, Xuben Hou, Yingkai Zhang, Benjamin G Neel, John P O'Bryan, Shohei Koide.

Activating mutants of RAS are commonly found in human cancers, but to date selective targeting of RAS in the clinic has been limited to KRAS(G12C) through covalent inhibitors. Here, we report a monobody, termed 12VC1, that recognizes the active state of both KRAS(G12V) and KRAS(G12C) up to 400-times more tightly than wild-type KRAS. The crystal structures reveal that 12VC1 recognizes the mutations through a shallow pocket, and 12VC1 competes against RAS-effector interaction. When expressed intracellularly, 12VC1 potently inhibits ERK activation and the proliferation of RAS-driven cancer cell lines in vitro and in mouse xenograft models. 12VC1 fused to VHL selectively degrades the KRAS mutants and provides more extended suppression of mutant RAS activity than inhibition by 12VC1 alone. These results demonstrate the feasibility of selective targeting and degradation of KRAS mutants in the active state with noncovalent reagents and provide a starting point for designing noncovalent therapeutics against oncogenic RAS mutants.

DOI: https://doi.org/10.1038/s41467-021-22969-5
Methods Mol Biol. 2021 ;2262 323-334

A Facile Method to Engineer Mutant Kras Alleles in an Isogenic Cell Background.

Konstantin Budagyan, Jonathan Chernoff.

  Oncogenic KRAS mutations are common in colorectal cancer (CRC), found in ~50% of tumors, and are associated with poor prognosis and resistance to therapy. There is substantial diversity of KRAS mutations observed in CRC. Importantly, emerging clinical and experimental analysis of relatively common KRAS mutations at amino acids G12, G13, A146, and Q61 suggest that each mutation differently influences the clinical properties of a disease and response to therapy. Although clinical evidence suggests biological differences between mutant KRAS alleles, these differences and the mechanisms underlying them are not well understood, and further exploration of allele-specific differences may provide evidence for individualized therapeutics. One approach to study allelic variation involves the use of isogenic cell lines that express different endogenous KRAS mutants. Here we developed an assay using fluorescent co-selection for CRISPR-driven gene editing to generate various Kras mutations in an isogenic murine colon epithelial cell line background. This assay involves generation of a cell line stably expressing Cas9 linked to BFP and simultaneous introduction of single-guide RNAs (sgRNAs) to two different gene loci resulting in double-editing events. Single-stranded donor oligonucleotides are introduced for a GFP gene and a Kras mutant allele of our choice as templates for homologous recombination (HDR). Cells that successfully undergo HDR are GFP-positive and have a higher probability of containing the desired Kras mutation. Therefore, selection for GFP-positive cells allows us to identify those with phenotypically silent Kras edits. Ultimately, this method allows us to toggle between different mutant alleles and preserve the wild-type allele while maintaining an isogenic background.

Keywords:  Cancer; Co-selection; Epithelial cells; Gene editing; Isogenic cells; KRAS; Signal transduction; Small G proteins

DOI:  https://doi.org/10.1007/978-1-0716-1190-6_20
Adv Exp Med Biol. 2021 ;1269 169-177

The Warburg Effect: Historical Dogma Versus Current Rationale.

Peter Vaupel, Gabriele Multhoff.

  Contrary to Warburg's original thesis, accelerated aerobic glycolysis is not a primary and permanent consequence of dysfunctional mitochondria compensating for a poor ATP yield per mole glucose. Instead, the Warburg effect is an essential part of a "selfish" metabolic reprogramming, which results from the interplay between (normoxic or hypoxic) HIF-1 overexpression, oncogene activation (cMyc, Ras), loss of function of tumor suppressors (mutant p53, mutant PTEN, microRNAs and sirtuins with suppressor functions), activated (PI3K/Akt/mTORC1, Ras/Raf/Mek/Erk/c-Myc) or deactivated (AMPK) signaling pathways, components of the tumor microenvironment, and HIF-1 cooperations with epigenetic mechanisms. Molecular and functional processes of the Warburg effect include (a) considerably accelerated glycolytic fluxes; (b) adequate ATP generation per unit time to maintain energy homeostasis; (c) backup and diversion of glycolytic intermediates facilitating the biosynthesis of nucleotides, nonessential amino acids, lipids, and hexosamines; (d) inhibition of pyruvate entry into mitochondria; (e) excessive formation and accumulation of lactate which stimulates tumor growth and suppression of antitumor immunity (in addition, lactate can serve as an energy source for normoxic cancer cells, contributes to extracellular acidosis, and thus drives malignant progression and resistances to conventional therapies); (f) maintenance of the cellular redox homeostasis and low ROS formation; and (g) HIF-1 overexpression, mutant p53, and mutant PTEN which inhibit mitochondrial biogenesis and functions, thus negatively impacting cellular respiration rate. The glycolytic switch is an early event in oncogenesis and primarily supports cell survival. All in all, the Warburg effect, i.e., aerobic glycolysis in the presence of oxygen and - in principle - functioning mitochondria, constitutes a major driver of the cancer progression machinery, resistance to conventional therapies, and - finally - poor patient outcome.

Keywords:  ATP generation; Aerobic glycolysis; Biosynthesis of macromolecules; Energy homeostasis; Glycolytic phenotype; Glycolytic switch; Lactate accumulation; Metabolic reprogramming; Oncogenesis; Redox homeostasis; Tumor acidosis; Tumor glucose metabolism; Tumor mitochondria; Warburg effect

DOI:  https://doi.org/10.1007/978-3-030-48238-1_27
Methods Mol Biol. 2021 ;2262 281-302

Probing RAS Function with Monobodies.

Imran Khan, John P O'Bryan.

  RAS is frequently mutated in human cancers with nearly 20% of all cancers harboring mutations in one of three RAS isoforms (KRAS, HRAS, or NRAS). Furthermore, RAS proteins are critical oncogenic drivers of tumorigenesis. As such, RAS has been a prime focus for development of targeted cancer therapeutics. Although RAS is viewed by many as undruggable, the recent development of allele-specific covalent inhibitors to KRAS(G12C) has provided significant hope for the eventual pharmacological inhibition of RAS (Ostrem et al., Nature 503(7477):548-551, 2013; Patricelli et al., Cancer Discov 6(3):316-329, 2016; Janes et al., Cell 172(3):578-589.e17, 2018; Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Indeed, these (G12C)-specific inhibitors have elicited promising responses in early phase clinical trials (Canon et al., Nature 575(7781):217-223, 2019; Hallin et al., Cancer Discov 10(1):54-71, 2020). Despite this success in pharmacologically targeting KRAS(G12C), the remaining RAS mutants lack readily tractable chemistries for development of covalent inhibitors. Thus, alternative approaches are needed to develop broadly efficacious RAS inhibitors. We have utilized Monobody (Mb) technology to identify vulnerabilities in RAS that can potentially be exploited for development of novel RAS inhibitors. Here, we describe the methods used to isolate RAS-specific Mbs and to define their inhibitory activity.

Keywords:  Cell signaling; HEK293; Monobody; NIH/3T3; PEI; RAS; RAS foci; Soft agar assays; Transfection; Tumorigenesis; Xenograft tumor assays

DOI:  https://doi.org/10.1007/978-1-0716-1190-6_17
Methods Mol Biol. 2021 ;2262 349-360

Generation of Patient-Derived Colorectal Cancer Organoids for RAS Studies.

Dirk Schumacher, Joseph L Regan, Dorothea Przybilla, Reinhold Schäfer.

  Human cell line models have been widely used for testing of novel anticancer compounds and for predicting clinical response to monotherapies and combinatorial therapies. For many years, standard monolayer culture conditions were used as gold standard, only surpassed by in vivo testing of mouse models. Recently, the incorporation of three-dimensional culture has been shown to further improve predictive compound testing. In view of the renewed interest in anti-RAS cancer therapy, we provide a protocol for establishing colorectal cancer organoids which are characterized by a high prevalence of KRAS mutations.

Keywords:  Colorectal cancer; Immunofluorescence; Immunohistochemistry; Marker expression; Preclinical drug testing; Three-dimensional culture

DOI:  https://doi.org/10.1007/978-1-0716-1190-6_22
Biochem Biophys Res Commun. 2021 May 11. pii: S0006-291X(21)00756-7. [Epub ahead of print]560 59-65

The association between ERK inhibitor sensitivity and molecular characteristics in colorectal cancer.

Hodaka Tayama, Hideaki Karasawa, Akihiro Yamamura, Yasunobu Okamura, Fumiki Katsuoka, Hideyuki Suzuki, Taiki Kajiwara, Minoru Kobayashi, Yuuri Hatsuzawa, Masahiro Shiihara, Li Bin, Md Yeashin Gazi, Mizuki Sato, Kazuki Kumada, Shigehiro Ito, Muneaki Shimada, Toru Furukawa, Takashi Kamei, Shinobu Ohnuma, Michiaki Unno.

  The mitogen-activated protein kinase (MAPK) pathway plays an important role in the colorectal cancer (CRC) progression, being supposed to be activated by the gene mutations, such as BRAF or KRAS. Although the inhibitors of extracellular signal-regulated kinase (ERK) have demonstrated efficacy in the cells with the BRAF or KRAS mutations, a clinical response is not always associated with the molecular signature. The patient-derived organoids (PDO) have emerged as a powerful in vitro model system to study cancer, and it has been widely applied for the drug screening. The present study aims to analyze the association between the molecular characteristics which analyzed by next-generation sequencing (NGS) and sensitivity to the ERK inhibitor (i.e., SCH772984) in PDO derived from CRC specimens. A drug sensitivity test for the SCH772984 was conducted using 14 CRC cell lines, and the results demonstrated that the sensitivity was in agreement with the BRAF mutation, but was not completely consistent with the KRAS status. In the drug sensitivity test for PDO, 6 out of 7 cases with either BRAF or KRAS mutations showed sensitivity to the SCH772984, while 5 out of 6 cases of both BRAF and KRAS wild-types were resistant. The results of this study suggested that the molecular status of the clinical specimens are likely to represent the sensitivity in the PDOs but is not necessarily absolutely overlapping. PDO might be able to complement the limitations of the gene panel and have the potential to provide a novel precision medicine.

Keywords:  ERK inhibitor; MAPK; NGS; PDO

DOI:  https://doi.org/10.1016/j.bbrc.2021.04.130
Methods Mol Biol. 2021 ;2262 311-321

Mathematical Modeling to Study KRAS Mutant-Specific Responses to Pathway Inhibition.

Edward C Stites.

  This chapter will describe how mathematical modeling allows the RAS pathway to be studied with computational experiments. The mathematical model utilized simulates the biochemical reactions that regulate RAS signaling. This type of model incorporates knowledge of reaction mechanisms, including measured quantitative parameters that characterize these reactions for both wild-type and mutant RAS proteins. For an illustrative example, this chapter focuses on how modeling provided new insights that helped solve a problem that challenged the RAS community for nearly a decade: why do colorectal cancers with the KRAS G13D mutation, but not the other common KRAS mutations, benefit from EGFR inhibition? The methods described include computational dose-response experiments and the use of "computational chimeric" RAS mutants.

Keywords:  Computational biology; Systems biology; Systems pharmacology; Targeted therapy

DOI:  https://doi.org/10.1007/978-1-0716-1190-6_19
J Cancer. 2021 ;12(11): 3154-3163

MiRNA-202-5p promotes Colorectal Carcinogenesis through suppression of PTEN.

Jing Huang, Yaqin Zhang, Yuan Xu, Qi Xie, Shuang Wu, Yi Dong, Bing Chen, Yang Xia, Lili Guo, Qun Li, Hao Gu, Wanglai Hu.

  Colorectal cancer (CRC) is still one of the leading causes of cancer-associated death in the modern society. The biological function of miR-202-5p for CRC development remains controversial, largely due to the fact that miR-202-5p can be tumor-suppressive or oncogenic in different contexts. Obtained results indicated that aberrant expression of miR-202-5p was observed in majority of human CRC samples and miR-202-5p was transcriptionally up-regulated by c-Myc. In addition, miR-202-5p functions to promote the activation of PI3K/Akt signaling pathway by directly suppressing PTEN. Silencing or enforced expression of miR-202-5p resulted in CRC cell proliferation inhibition and enhancement, respectively. Importantly, decreased PTEN level and increased phosphorylation of Akt were frequently associated with elevated miR-202-5p expression in colorectal cancer tissues. Increased miR-202-5p expression may serve as a tumor promoter by directly targeting PTEN in colorectal cancer.

Keywords:  CRC; PTEN; c-Myc; miR-202-5p

DOI:  https://doi.org/10.7150/jca.56186
Cancer Sci. 2021 May 02.

Specific activation of glycolytic enzyme enolase 2 in BRAF V600E-mutated colorectal cancer.

Ryohei Yukimoto, Naohiro Nishida, Tsuyoshi Hata, Shiki Fujino, Takayuki Ogino, Norikatsu Miyoshi, Hidekazu Takahashi, Mamoru Uemura, Taroh Satoh, Yamamoto Hirofumi, Tsunekazu Mizushima, Yuichiro Doki, Hidetoshi Eguchi.

  The BRAF V600E mutation occurs in approximately 10% of patients with metastatic colorectal cancer (CRC) and constitutes a distinct subtype of the disease with extremely poor prognosis. To address this refractory disease, we investigated the unique metabolic gene profile of BRAF V600E-mutated tumors via in silico analysis using a large-scale clinical database. We found that BRAF V600E-mutated tumors exhibited a specific metabolic gene expression signature, including some genes that are associated with poor prognosis in CRC. We discovered that BRAF V600E-mutated tumors expressed high levels of glycolytic enzyme enolase 2 (ENO2), which is mainly expressed in neuronal tissues under physiological conditions. In vitro experiments using CRC cells demonstrated that BRAF V600E-mutated cells exhibited enhanced dependency on ENO2 compared to BRAF wild-type cancer cells and that knockdown of ENO2 led to the inhibition of proliferation and migration of BRAF V600E-mutated cancer cells. Moreover, inhibition of ENO2 resulted in enhanced sensitivity to vemurafenib, a selective inhibitor of BRAF V600E. We identified AP-1 transcription factor subunit (FOSL1) as being involved in the transcription of ENO2 in CRC cells. In addition, both MAPK and PI3K/Akt signaling were suppressed upon inhibition of ENO2, implying an additional oncogenic role of ENO2. These results suggest the crucial role of ENO2 in the progression of BRAF V600E-mutated CRC and indicate the therapeutic implications of targeting this gene.

Keywords:  BRAF V600E-mutated colorectal cancer; ENO2; FOSL1; glycolysis; metabolic genes

DOI:  https://doi.org/10.1111/cas.14929
Cancer Res. 2021 May 12. pii: canres.2496.2020. [Epub ahead of print]

Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition.

George Sharbeen, Joshua A McCarroll, Anouschka Akerman, Chantal Kopecky, Janet Youkhana, John Kokkinos, Jeff Holst, Cyrille Boyer, Mert Erkan, David Goldstein, Paul Timpson, Thomas R Cox, Brooke A Pereira, Jessica L Chitty, Sigrid K Fey, Arafath K Najumudeen, Andrew D Campbell, Owen J Sansom, Rosa Mistica C Ignacio, Stephanie Naim, Jie Liu, Nelson Russia, Julia Lee, Angela Chou, Amber Johns, Anthony J Gill, Estrella Gonzales-Aloy, Val Gebski, Yi Fang Guan, Marina Pajic, Nigel Turner, Minoti V Apte, Thomas P Davis, Jennifer P Morton, Koroush S Haghighi, Jorjina Kasparian, Benjamin J McLean, Yordanos F I Setargew, Australian Pancreatic Cancer Genome Initiative Apgi, Phoebe A Phillips.

Cancer-Associated Fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through pro-tumor signaling and the generation of fibrosis that creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11, a cystine transporter, has been identified as a potential therapeutic target in PDAC cells. However, the role of SLC7A11 in PDAC tumor stroma and its prognostic significance has not been evaluated. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poor overall survival. Orthogonal approaches showed that PDAC-derived CAFs were dependent on SLC7A11 for cystine uptake and glutathione synthesis. SLC7A11 inhibition significantly decreased CAF proliferation, reduced their resistance to oxidative stress, and inhibited their ability to remodel collagen and support PDAC cell growth. Specific ablation of SLC7A11 from the tumor compartment of transgenic mice did not affect PDAC growth, suggesting the stroma can substantially influence PDAC response to SLC7A11 inhibition. In an orthotopic PDAC mouse model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle SLC7A11-silencing drug developed by our laboratory reduced PDAC tumor growth, metastasis, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-2496
Front Cell Dev Biol. 2021 ;9 667311

Folliculin: A Regulator of Transcription Through AMPK and mTOR Signaling Pathways.

Josué M J Ramirez Reyes, Rafael Cuesta, Arnim Pause.

  Folliculin (FLCN) is a tumor suppressor gene responsible for the inherited Birt-Hogg-Dubé (BHD) syndrome, which affects kidneys, skin and lungs. FLCN is a highly conserved protein that forms a complex with folliculin interacting proteins 1 and 2 (FNIP1/2). Although its sequence does not show homology to known functional domains, structural studies have determined a role of FLCN as a GTPase activating protein (GAP) for small GTPases such as Rag GTPases. FLCN GAP activity on the Rags is required for the recruitment of mTORC1 and the transcriptional factors TFEB and TFE3 on the lysosome, where mTORC1 phosphorylates and inactivates these factors. TFEB/TFE3 are master regulators of lysosomal biogenesis and function, and autophagy. By this mechanism, FLCN/FNIP complex participates in the control of metabolic processes. AMPK, a key regulator of catabolism, interacts with FLCN/FNIP complex. FLCN loss results in constitutive activation of AMPK, which suggests an additional mechanism by which FLCN/FNIP may control metabolism. AMPK regulates the expression and activity of the transcriptional cofactors PGC1α/β, implicated in the control of mitochondrial biogenesis and oxidative metabolism. In this review, we summarize our current knowledge of the interplay between mTORC1, FLCN/FNIP, and AMPK and their implications in the control of cellular homeostasis through the transcriptional activity of TFEB/TFE3 and PGC1α/β. Other pathways and cellular processes regulated by FLCN will be briefly discussed.

Keywords:  AMPK; PGC1α; TFE3; TFEB; folliculin; mTORC1; metabolism; transcriptional regulation

DOI:  https://doi.org/10.3389/fcell.2021.667311
Front Mol Biosci. 2021 ;8 662579

Advances and Insights of APC-Asef Inhibitors for Metastatic Colorectal Cancer Therapy.

Xiuyan Yang, Jie Zhong, Qiufen Zhang, Li Feng, Zhen Zheng, Jian Zhang, Shaoyong Lu.

  In Colorectal cancer (CRC), adenomatous polyposis coli (APC) directly interacts with the Rho guanine nucleotide exchange factor 4 (Asef) and releases its GEF activity. Activated Asef promotes the aberrant migration and invasion of CRC cell through a CDC42-mediated pathway. Knockdown of either APC or Asef significantly decreases the migration of CRC cells. Therefore, disrupting the APC-Asef interaction is a promising strategy for the treatment of invasive CRC. With the growth of structural information, APC-Asef inhibitors have been designed, providing hope for CRC therapy. Here, we will review the APC-Asef interaction in cancer biology, the structural complex of APC-Asef, two generations of peptide inhibitors of APC-Asef, and small molecule inhibitors of APC-Asef, focusing on research articles over the past 30 years. We posit that these advances in the discovery of APC-Asef inhibitors establish the protein-protein interaction (PPI) as targetable and provide a framework for other PPI programs.

Keywords:  APC-Asef inhibitors; colorectal cancer therapy; peptide inhibitors; small molecule inhibitors; structure based drug design

DOI:  https://doi.org/10.3389/fmolb.2021.662579
EMBO J. 2021 May 14. e106412

ArfGAP1 inhibits mTORC1 lysosomal localization and activation.

Delong Meng, Qianmei Yang, Chase H Melick, Brenden C Park, Ting-Sung Hsieh, Adna Curukovic, Mi-Hyeon Jeong, Junmei Zhang, Nicholas G James, Jenna L Jewell.

  The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid-deficient conditions is not completely understood. Here, we identify ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature-sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.

Keywords:  ArfGAP1; amino acids; lysosome; mTORC1; vesicle trafficking

DOI:  https://doi.org/10.15252/embj.2020106412
Bioorg Med Chem. 2021 May 01. pii: S0968-0896(21)00198-X. [Epub ahead of print]40 116190

The chemistry and biology of phosphatidylinositol 4-phosphate at the plasma membrane.

Alex G Batrouni, Jeremy M Baskin.

  Phosphoinositides are an important class of anionic, low abundance signaling lipids distributed throughout intracellular membranes. The plasma membrane contains three phosphoinositides: PI(4)P, PI(4,5)P2, and PI(3,4,5)P3. Of these, PI(4)P has remained the most mysterious, despite its characterization in this membrane more than a half-century ago. Fortunately, recent methodological innovations at the chemistry-biology interface have spurred a renaissance of interest in PI(4)P. Here, we describe these new toolsets and how they have revealed novel functions for the plasma membrane PI(4)P pool. We examine high-resolution structural characterization of the plasma membrane PI 4-kinase complex that produces PI(4)P, tools for modulating PI(4)P levels including isoform-selective PI 4-kinase inhibitors, and fluorescent probes for visualizing PI(4)P. Collectively, these chemical and biochemical approaches have revealed insights into how cells regulate synthesis of PI(4)P and its downstream metabolites as well as new roles for plasma membrane PI(4)P in non-vesicular lipid transport, membrane homeostasis and trafficking, and cell signaling pathways.

Keywords:  GPCR signaling; Genetically encoded biosensors; Lipid kinase inhibitors; PI(4)P; PI4KIIIα; Phosphoinositides; Phospholipid transport

DOI:  https://doi.org/10.1016/j.bmc.2021.116190
Methods Mol Biol. 2021 ;2262 91-103

Validation of Isoform- and Mutation-Specific RAS Antibodies.

Andrew M Waters, Channing J Der.

  Validation of antibody specificity is essential for the accurate evaluation of protein expression. For antibodies that recognize the gene products of the RAS family of oncogenes (HRAS, KRAS, and NRAS), an important challenge is the determination of selectivity for the four nearly identical HRAS, KRAS4A, KRAS4B, and NRAS proteins. With increasing appreciation for the distinct roles of the different RAS proteins in normal and neoplastic cells, there is a need for well-validated antibodies to evaluate the function and expression of the different RAS isoforms. Here we describe our experimental approaches to characterize RAS antibodies for their isoform- and mutant-specificity for use in immunoblot analyses.

Keywords:  Antibodies; HRAS; Immunoblots; KRAS; MEFs; Mouse embryonic fibroblasts; NRAS; RAS; Western blots

DOI:  https://doi.org/10.1007/978-1-0716-1190-6_5
EMBO Rep. 2021 May 13. e52173

OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring.

Biao Wang, Wei He, Philipp P Prosseda, Liang Li, Tia J Kowal, Jorge A Alvarado, Qing Wang, Yang Hu, Yang Sun.

  Lysosomal positioning and mTOR (mammalian target of rapamycin) signaling coordinate cellular responses to nutrient levels. Inadequate nutrient sensing can result in growth delays, a hallmark of Lowe syndrome. OCRL mutations cause Lowe syndrome, but the role of OCRL in nutrient sensing is unknown. Here, we show that OCRL is localized to the centrosome by its ASH domain and that it recruits microtubule-anchoring factor SSX2IP to the centrosome, which is important in the formation of the microtubule-organizing center. Deficiency of OCRL in human and mouse cells results in loss of microtubule-organizing centers and impaired microtubule-based lysosome movement, which in turn leads to mTORC1 inactivation and abnormal nutrient sensing. Centrosome-targeted PACT-SSX2IP can restore microtubule anchoring and mTOR activity. Importantly, boosting the activity of mTORC1 restores the nutrient sensing ability of Lowe patients' cells. Our findings highlight mTORC1 as a novel therapeutic target for Lowe syndrome.

Keywords:  OCRL; lowe syndrome; lysosome positioning; mTOR; microtubule nucleation

DOI:  https://doi.org/10.15252/embr.202052173
Expert Opin Ther Pat. 2021 May 10.

Phosphatidylinositol 3-kinase (PI3K) inhibitors: a recent update on inhibitor design and clinical trials (2016-2020).

Dima A Sabbah, Rima Hajjo, Sanaa K Bardaweel, Haizhen A Zhong.

   INTRODUCTION: The phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway plays a central role in regulating cell growth and proliferation and thus have been considered as effective anticancer drug targets. Many PI3K inhibitors have been developed and progressed to various stages of clinical trials and some even have been approved in recent years by the regulatory agency as anticancer treatment. In this review, we discuss the drug design and clinical development of PI3K inhibitors in the past four years. We review in details the selectivity and potency of forty-seven PI3K inhibitors, classified based on the mechanism of action. Structural determinants for increasing selectivity toward PI3K subtype-selectivity or mutant selectivity are discussed. Future research direction and current clinical development in combination therapy of inhibitors involving in the PI3Ks are also discussed.Area covered: This review covers clinical trial reports and patent literature on PI3K inhibitors and their selectivity published between 2016 and 2020.Expert opinion: To address the gain-of-function of PI3Kα mutants (E542K, E545K, and H1047R), it is highly desirable to design and develop mutant-specific PI3K inhibitors. It is also necessary to develop subtype-selective PI3Kα inhibitors to minimize toxicity. To reduce drug resistance and to improve efficacy, future studies should include combination therapy of PI3K inhibitors with existing anticancer drugs from different pathways.

Keywords:  PI3K; cancer; clinical trials; combination therapy; inhibitors; trafficking cascade

DOI:  https://doi.org/10.1080/13543776.2021.1924150
J Control Release. 2021 Apr 30. pii: S0168-3659(21)00208-X. [Epub ahead of print]334 335-352

A synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3'-phosphatase (PNKP) for targeted therapy of PTEN-deficient colorectal cancer.

Sams M A Sadat, Igor M Paiva, Zahra Shire, Forughalsadat Sanaee, Timothy D R Morgan, Marco Paladino, Feridoun Karimi-Busheri, Rajam S Mani, Gary R Martin, Frank R Jirik, Dennis G Hall, Michael Weinfeld, Afsaneh Lavasanifar.

  Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN-/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.

Keywords:  CETSA; Colorectal cancer; Nanoparticle; PNKP; PTEN; Synthetic lethality

DOI:  https://doi.org/10.1016/j.jconrel.2021.04.034
Biochim Biophys Acta Mol Cell Res. 2021 May 11. pii: S0167-4889(21)00113-0. [Epub ahead of print] 119059

The role of GSK3 in metabolic pathway perturbations in cancer.

David Papadopoli, Michael Pollak, Ivan Topisirovic.

  Malignant transformation and tumor progression are accompanied by significant perturbations in metabolic programs. As such, cancer cells support high ATP turnover to construct the building blocks needed to fuel neoplastic growth. The coordination of metabolic networks in malignant cells is dependent on the collaboration with cellular signaling pathways. Glycogen synthase kinase 3 (GSK3) lies at the convergence of several signaling axes, including the PI3K/AKT/mTOR, AMPK, and Wnt pathways, which influence cancer initiation, progression and therapeutic responses. Accordingly, GSK3 modulates metabolic processes, including protein and lipid synthesis, glucose, and mitochondrial metabolism, as well as autophagy. In this review, we highlight current knowledge of the role of GSK3 in metabolic perturbations in cancer.

Keywords:  AMPK; Cancer; GSK3; Metabolism; mTOR

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119059
Cell Signal. 2021 Apr 30. pii: S0898-6568(21)00123-6. [Epub ahead of print]84 110034

Cell-cell and cell-substratum contacts in the regulation of MAPK and Akt signalling: Importance in therapy, biopharmacy and bioproduction.

Aurimas Stulpinas, Tomas Uzusienis, Ausra Imbrasaite, Natalija Krestnikova, Ausra Unguryte, Audrone V Kalvelyte.

  The use of cultured cells as a tool for research, precision medicine, biopharmacy, and biomanufacturing is constantly increasing. In parallel, the role of cell-cell and cell-substratum contacts in cell functioning is increasingly validated. Adhesion signalling plays a key role here. The activity of cell fate-regulating signalling molecules is an important factor in determining cell behaviour, as well as their response to treatment, depending on cell phenotypic status and location in the body. Three cellular state models (adherent, single cells in suspension, and aggregated cells) were compared for cell signalling, including focal adhesion (FAK), mitogen-activated (MAPK), as well as Akt protein kinases, and transcription factor cJun, by using lung adenocarcinoma A549, muscle-derived stem Myo, as well as primary lung cancer cell lines. Survival of both A549 and Myo cells was dependent on kinases Akt and ERK in detached conditions. Intercellular contacts in aggregates promoted activation of Akt and ERK, and cell survival. Loss of contacts with the substrate increased phosphorylation of MAP kinases JNK and p38, while decreased Akt phosphorylation by processes involving FAK. Unexpectedly, detachment increased phosphorylation of antiapoptotic kinase ERK in A549, while in Myo stem cells ERK phosphorylation was downregulated. JNK target transcription factor cJun protein level was markedly diminished by contacts between cells possibly involving mechanism of proteasomal degradation. Furthermore, studies revealed the opposite dependence of molecules of the same signalling pathway - phospho-cJun and phospho-JNK - on cell culture density. Differences in ERK activation under detachment conditions indicate that targeting of prosurvival kinases during anoikis should be different in different cells. Moreover, the outcome of JNK activation in cells may depend on the amount of cJun, which is determined by cell-cell contacts.

Keywords:  Anoikis; Extracellular contacts; Focal adhesion kinase (FAK); Mitogen-activated protein kinases (MAPKs); Protein kinase Akt; Transcription factor AP-1/cJun

DOI:  https://doi.org/10.1016/j.cellsig.2021.110034
Methods Mol Biol. 2021 ;2262 271-280

Using BioID to Characterize the RAS Interactome.

Hema Adhikari, Christopher M Counter.

  Identifying the proteins that associate with RAS oncoproteins has great potential, not only to elucidate how these mutant proteins are regulated and signal but also to identify potential therapeutic targets. Here we describe a detailed protocol to employ proximity labeling by the BioID methodology, which has the advantage of capturing weak or transient interactions, to identify in an unbiased manner those proteins within the immediate vicinity of oncogenic RAS proteins.

Keywords:  BioID; BirA; HRAS; Interactome; KRAS; NRAS; Proximity labeling

DOI:  https://doi.org/10.1007/978-1-0716-1190-6_16
Front Oncol. 2021 ;11 650360

DDX54 Plays a Cancerous Role Through Activating P65 and AKT Signaling Pathway in Colorectal Cancer.

Yi Yu, Jing-Long Wang, Li-Li Meng, Chun-Ting Hu, Zhao-Wen Yan, Zhi-Ping He, Xiao-Qin Shi, Guo-Hui Fu, Li-Dong Zu.

  Colorectal cancer (CRC) is one of the most malignant cancers, and its incidence is still steadily increasing. The DDX RNA helicase family members have been found to play a role in various cancers; however, the role of DDX54 in colorectal cancer is still unclear and needed to be defined. Here, we found DDX54 was overexpressed in CRC tissues by the label-free mass spectrum, which was also verified in tissue microarray of colon cancer, as well as the CRC cell lines and TCGA database. High DDX54 level was correlated with tumor stage and distant metastasis, which always indicated a poor prognosis to the CRC patients. DDX54 could promote the proliferation and mobility of CRC cells through increasing the phosphorylation level p65 and AKT leading to the tumorigenesis. Here, we have preliminarily studied the function of DDX54 in CRC, which would improve our understanding of the underlying biology of CRC and provide the new insight that could be translated into novel therapeutic approaches.

Keywords:  AKT; DDX54; colorectal cancer; p65; proteomics

DOI:  https://doi.org/10.3389/fonc.2021.650360
Methods Mol Biol. 2021 ;2262 47-64

Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry.

Lauren M Adams, Caroline J DeHart, Neil L Kelleher.

  The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.

Keywords:  Immunoprecipitation; Intact protein; Isoform; Proteoform; RAS; Top-down mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-1190-6_3
Cancer Res. 2021 May 13. pii: canres.3694.2020. [Epub ahead of print]

SLFN5 regulates LAT1-mediated mTOR activation in castration-resistant prostate cancer.

Rafael S Martinez, Mark J Salji, Linda Rushworth, Chara Ntala, Giovanny Rodriguez Blanco, Ann Hedley, William Clark, Paul Peixoto, Eric Hervouet, Elodie Renaude, Sonia H Y Kung, Laura C A Galbraith, Colin Nixon, Sergio Lilla, Gillian M Mackay, Ladan Fazli, Luke Gaughan, David Sumpton, Martin E Gleave, Sara Zanivan, Arnaud Blomme, Hing Y Leung.

Androgen-deprivation therapy (ADT) is the standard of care for treatment of non-resectable prostate cancer (PCa). Despite high treatment efficiency, most patients ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we performed a comparative proteomic analysis of three in vivo, androgen receptor (AR)-responsive orthograft models of matched hormone-naive PCa and CRPC. Differential proteomic analysis revealed that distinct molecular mechanisms, including amino acid (AA) and fatty acid (FA) metabolism, are involved in the response to ADT in the different models. Despite this heterogeneity, Schlafen family member 5 (SLFN5) was identified as an AR-regulated protein in CRPC. SLFN5 expression was high in CRPC tumors and correlated with poor patient outcome. In vivo, SLFN5 depletion strongly impaired tumor growth in castrated conditions. Mechanistically, SLFN5 interacted with ATF4 and regulated the expression of LAT1, an essential AA transporter. Consequently, SLFN5 depletion in CRPC cells decreased intracellular levels of essential AA and impaired mTORC1 signalling in a LAT1-dependent manner. These results confirm that these orthograft models recapitulate the high degree of heterogeneity observed in CRPC patients and further highlight SLFN5 as a clinically relevant target for CRPC.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-3694