bims-pimaco 2022-11-13 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–11–13
sixteen papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

PTEN Loss Enhances Error-Prone DSB Processing and Tumor Cell Radiosensitivity by Suppressing RAD51 Expression and Homologous Recombination.
RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.
Mutations in the α4-α5 allosteric lobe of RAS do not significantly impair RAS signaling or self-association.
RAS: Circuitry and therapeutic targeting.
Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα.
The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner.
An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss.
Molecular characterization of acquired resistance to KRAS G12C-EGFR inhibition in colorectal cancer.
Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-mutant Pancreatic Cancer.
PI(4,5)P2: signaling the plasma membrane.
Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of L-type amino acid transporter 1.
Multiple congenital malformations arise from somatic mosaicism for constitutively active Pik3ca signaling.
The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult mouse gut.
Axin1 protects colon carcinogenesis by an immune-mediated effect.
Rac1 promotes the reprogramming of glucose metabolism and the growth of colon cancer cells through up-regulating SOX9.
mTOR inhibition amplifies the anti-lymphoma effect of PI3Kβ/δ blockage in diffuse large B-cell lymphoma.

Int J Mol Sci. 2022 Oct 25. pii: 12876. [Epub ahead of print]23(21):

PTEN Loss Enhances Error-Prone DSB Processing and Tumor Cell Radiosensitivity by Suppressing RAD51 Expression and Homologous Recombination.

Xile Pei, Emil Mladenov, Aashish Soni, Fanghua Li, Martin Stuschke, George Iliakis.

  PTEN has been implicated in the repair of DNA double-strand breaks (DSBs), particularly through homologous recombination (HR). However, other data fail to demonstrate a direct role of PTEN in DSB repair. Therefore, here, we report experiments designed to further investigate the role of PTEN in DSB repair. We emphasize the consequences of PTEN loss in the engagement of the four DSB repair pathways-classical non-homologous end-joining (c-NHEJ), HR, alternative end-joining (alt-EJ) and single strand annealing (SSA)-and analyze the resulting dynamic changes in their utilization. We quantitate the effect of PTEN knockdown on cell radiosensitivity to killing, as well as checkpoint responses in normal and tumor cell lines. We find that disruption of PTEN sensitizes cells to ionizing radiation (IR). This radiosensitization is associated with a reduction in RAD51 expression that compromises HR and causes a marked increase in SSA engagement, an error-prone DSB repair pathway, while alt-EJ and c-NHEJ remain unchanged after PTEN knockdown. The G2-checkpoint is partially suppressed after PTEN knockdown, corroborating the associated HR suppression. Notably, PTEN deficiency radiosensitizes cells to PARP inhibitors, Olaparib and BMN673. The results show the crucial role of PTEN in DSB repair and show a molecular link between PTEN and HR through the regulation of RAD51 expression. The expected benefit from combination treatment with Olaparib or BMN673 and IR shows that PTEN status may also be useful for patient stratification in clinical treatment protocols combining IR with PARP inhibitors.

Keywords:  DDR; DSBs; HR; PARP inhibitors; PTEN; SSA; alt-EJ; c-NHEJ; ionizing radiation

DOI:  https://doi.org/10.3390/ijms232112876
Mol Cell. 2022 Nov 02. pii: S1097-2765(22)01016-4. [Epub ahead of print]

RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.

Russell Spencer-Smith, Elizabeth M Terrell, Christine Insinna, Constance Agamasu, Morgan E Wagner, Daniel A Ritt, Jim Stauffer, Andrew G Stephen, Deborah K Morrison.

  BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

Keywords:  BRAF; CRAF; CRD; RAF kinases; RAS; RASopathies; autoinhibition; cysteine-rich domain; development; phosphatidylserine

DOI:  https://doi.org/10.1016/j.molcel.2022.10.016
J Biol Chem. 2022 Nov 02. pii: S0021-9258(22)01104-8. [Epub ahead of print] 102661

Mutations in the α4-α5 allosteric lobe of RAS do not significantly impair RAS signaling or self-association.

Michael Whaby, Lauren Wallon, Megan Mazzei, Imran Khan, Kai Wen Teng, Shohei Koide, John P O'Bryan.

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors. The α4-α5 allosteric lobe of RAS has been proposed as a RAS dimerization interface. Indeed, the NS1 monobody, which binds the α4-α5 region within the RAS G domain, inhibits RAS-dependent signaling and transformation as well as RAS nanoclustering at the plasma membrane. Although these results are consistent with a model in which the G domain dimerizes through the α4-α5 region, the isolated G domain of RAS lacks intrinsic dimerization capacity. Furthermore, prior studies analyzing α4-α5 point mutations have reported mixed effects on RAS function. Here, we evaluated the activity of a panel of single amino acid substitutions in the α4-α5 region implicated in RAS dimerization. We found that these proposed "dimerization-disrupting" mutations do not significantly impair self-association, signaling, or transformation of oncogenic RAS. These results are consistent with a model in which activated RAS protomers cluster in close proximity to promote the dimerization of their associated effector proteins (e.g., RAF) without physically associating into dimers mediated by specific molecular interactions. Our findings suggest the need for a nonconventional approach to developing therapeutics targeting the α4-α5 region.

DOI: https://doi.org/10.1016/j.jbc.2022.102661
Cell Signal. 2022 Oct 28. pii: S0898-6568(22)00267-4. [Epub ahead of print]101 110505

RAS: Circuitry and therapeutic targeting.

Gagandeep Singh, Neelam Thakur, Umesh Kumar.

  Cancer has affected the lives of millions worldwide and is truly regarded as a devastating disease process. Despite advanced understanding of the genomic underpinning of cancer development and progression, therapeutic challenges are still persistent. Among all the human cancers, around 33% are attributed to mutations in RAS oncogene, a crucial component of the signaling pathways. With time, our understanding of RAS circuitry has improved and now the fact that it activates several downstream effectors, depending on the type and grades of cancer has been established. The circuitry is controlled via post-transcriptional mechanisms and frequent distortions in these mechanisms lead to important metabolic as well as immunological states that favor cancer cells' growth, survival, plasticity and metastasis. Therefore, understanding RAS circuitry can help researchers/clinicians to develop novel and potent therapeutics that, in turn, can save the lives of patients suffering from RAS-mutant cancers. There are many challenges presented by resistance and the potential strategies with a particular focus on novel combinations for overcoming these, that could move beyond transitory responses in the direction of treatment. Here in this review, we will look at how understanding the circuitry of RAS can be put to use in making strategies for developing therapeutics against RAS- driven malignancies.

Keywords:  RAS; RAS circuitry; RAS dependent cancer

DOI:  https://doi.org/10.1016/j.cellsig.2022.110505
Proc Natl Acad Sci U S A. 2022 11 16. 119(46): e2215621119

Cryo-EM structures of cancer-specific helical and kinase domain mutations of PI3Kα.

Xiao Liu, Qingtong Zhou, Jonathan R Hart, Yingna Xu, Su Yang, Dehua Yang, Peter K Vogt, Ming-Wei Wang.

  Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that perform multiple and important cellular functions. The protein investigated here belongs to class IA of the PI3Ks; it is a dimer consisting of a catalytic subunit, p110α, and a regulatory subunit, p85α, and is referred to as PI3Kα. The catalytic subunit p110α is frequently mutated in cancer. The mutations induce a gain of function and constitute a driving force in cancer development. About 80% of these mutations lead to single-amino-acid substitutions in one of three sites of p110α: two in the helical domain of the protein (E542K and E545K) and one at the C-terminus of the kinase domain (H1047R). Here, we report the cryo-electron microscopy structures of these mutants in complex with the p110α-specific inhibitor BYL-719. The H1047R mutant rotates its sidechain to a new position and weakens the kα11 activation loop interaction, thereby reducing the inhibitory effect of p85α on p110α. E542K and E545K completely abolish the tight interaction between the helical domain of p110α and the N-terminal SH2 domain of p85α and lead to the disruption of all p85α binding and a dramatic increase in flexibility of the adaptor-binding domain (ABD) in p110α. Yet, the dimerization of PI3Kα is preserved through the ABD-p85α interaction. The local and global structural features induced by these mutations provide molecular insights into the activation of PI3Kα, deepen our understanding of the oncogenic mechanism of this important signaling molecule, and may facilitate the development of mutant-specific inhibitors.

Keywords:  mass spectrometry; mutants; phosphoinositide 3-kinase (PI3K)

DOI:  https://doi.org/10.1073/pnas.2215621119
EMBO J. 2022 Nov 10. e110833

The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner.

Belén Sanz-Castillo, Begoña Hurtado, Diana Vara-Ciruelos, Aicha El Bakkali, Dario Hermida, Beatriz Salvador-Barbero, Diego Martínez-Alonso, José González-Martínez, Clara Santiveri, Ramón Campos-Olivas, Pilar Ximénez-Embún, Javier Muñoz, Mónica Álvarez-Fernández, Marcos Malumbres.

  The AKT-mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However, how cells control the signals that limit AKT activity is not fully understood. Here, we show that MASTL/Greatwall, a cell cycle kinase that supports mitosis by phosphorylating the PP2A/B55 inhibitors ENSA/ARPP19, inhibits PI3K-AKT activity by sustaining mTORC1- and S6K1-dependent phosphorylation of IRS1 and GRB10. Genetic depletion of MASTL results in an inefficient feedback loop and AKT hyperactivity. These defects are rescued by the expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, thereby limiting the PP2A/B55-dependent dephosphorylation of IRS1 and GRB10 downstream of mTORC1. Downregulation of MASTL results in increased glucose uptake in vitro and increased glucose tolerance in adult mice, suggesting the relevance of the MASTL-PP2A/B55 kinase-phosphatase module in controlling AKT and maintaining metabolic homeostasis.

Keywords:  AKT; MASTL; cell cycle; glucose homeostasis; mTOR

DOI:  https://doi.org/10.15252/embj.2022110833
Nat Commun. 2022 Nov 10. 13(1): 6808

An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss.

Kaushal Asrani, Juhyung Woo, Adrianna A Mendes, Ethan Schaffer, Thiago Vidotto, Clarence Rachel Villanueva, Kewen Feng, Lia Oliveira, Sanjana Murali, Hans B Liu, Daniela C Salles, Brandon Lam, Pedram Argani, Tamara L Lotan.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.

DOI: https://doi.org/10.1038/s41467-022-34617-7
Cancer Discov. 2022 Nov 10. pii: CD-22-0405. [Epub ahead of print]

Molecular characterization of acquired resistance to KRAS G12C-EGFR inhibition in colorectal cancer.

Rona Yaeger, Riccardo Mezzadra, Jenna Sinopoli, Yu Bian, Michelangelo Marasco, Esther Kaplun, Yijun Gao, HuiYong Zhao, Arnaud Da Cruz Paula, Yingjie Zhu, Almudena Chaves Perez, Kalyani Chadalavada, Edison Tse, Sudhir Chowdhry, Sydney Bowker, Qing Chang, Besnik Qeriqi, Britta Weigelt, Gouri J Nanjangud, Michael F Berger, Hirak Der-Torossian, Kenna Anderes, Nicholas D Socci, Jinru Shia, Gregory J Riely, Yonina R Murciano-Goroff, Bob T Li, James G Christensen, Jorge S Reis-Filho, David B Solit, Elisa de Stanchina, Scott W Lowe, Neal Rosen, Sandra Misale.

With the combination of KRAS G12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRAS G12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRAS G12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance.

DOI: https://doi.org/10.1158/2159-8290.CD-22-0405
Cancer Res. 2022 Nov 08. pii: CAN-22-0391. [Epub ahead of print]

Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-mutant Pancreatic Cancer.

Craig M Goodwin, Andrew M Waters, Jennifer E Klomp, Sehrish Javaid, Kirsten L Bryant, Clint A Stalnecker, Kristina Drizyte-Miller, Bjoern Papke, Runying Yang, Amber M Amparo, Irem Ozkan-Dagliyan, Elisa Baldelli, Valerie Calvert, Mariaelena Pierobon, Jessica A Sorrentino, Andrew P Beelen, Natalie Bublitz, Mareen Lüthen, Kris C Wood, Emanuel F Petricoin, Christine Sers, Autumn J McRee, Adrienne D Cox, Channing J Der.

Mutational loss of CDKN2A (encoding p16INK4A) tumor suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, anti-apoptotic signaling, and MYC expression. Based on these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small molecule inhibitor-based approaches. Additionally, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to PDAC patients.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-0391
Biochem J. 2022 Nov 11. 479(21): 2311-2325

PI(4,5)P2: signaling the plasma membrane.

Rachel C Wills, Gerald R V Hammond.

  In the almost 70 years since the first hints of its existence, the phosphoinositide, phosphatidyl-D-myo-inositol 4,5-bisphosphate has been found to be central in the biological regulation of plasma membrane (PM) function. Here, we provide an overview of the signaling, transport and structural roles the lipid plays at the cell surface in animal cells. These include being substrate for second messenger generation, direct modulation of receptors, control of membrane traffic, regulation of ion channels and transporters, and modulation of the cytoskeleton and cell polarity. We conclude by re-evaluating PI(4,5)P2's designation as a signaling molecule, instead proposing a cofactor role, enabling PM-selective function for many proteins.

Keywords:  5P2; PIP2; PtdIns4; lipid rafts; phospholipids; signaling

DOI:  https://doi.org/10.1042/BCJ20220445
Cancer Metab. 2022 Nov 10. 10(1): 18

Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of L-type amino acid transporter 1.

Hiroki Okanishi, Ryuichi Ohgaki, Minhui Xu, Hitoshi Endou, Yoshikatsu Kanai.

   BACKGROUND: Cancer-upregulated L-type amino acid transporter 1 (LAT1; SLC7A5) supplies essential amino acids to cancer cells. LAT1 substrates are not only needed for cancer rapid growth, but involved in cellular signaling. LAT1 has been proposed as a potential target for cancer treatment-its inhibitor, JPH203, is currently in clinical trials and targets biliary tract cancer (BTC). Here, we revealed to what extent LAT1 inhibitor affects intracellular amino acid content and what kind of cellular signals are directly triggered by LAT1 inhibition.
METHODS: Liquid chromatography assay combined with o-phthalaldehyde- and 9-fluorenyl-methylchloroformate-based derivatization revealed changes in intracellular amino acid levels induced by LAT1 inhibition with JPH203 treatment in three BTC cell lines. Tandem mass tag-based quantitative phosphoproteomics characterized the effect of JPH203 treatment on BTC cells, and suggested key regulators in LAT1-inhibited cells. We further studied one of the key regulators, CK2 protein kinase, by using Western blot, enzymatic activity assay, and co-immunoprecipitation. We evaluated anticancer effects of combination of JPH203 with CK2 inhibitor using cell growth and would healing assay.
RESULTS: JPH203 treatment decreased intracellular levels of LAT1 substrates including essential amino acids of three BTC cell lines, immediately and drastically. We also found levels of some of these amino acids were partially recovered after longer-time treatment. Therefore, we performed phosphoproteomics with short-time JPH203 treatment prior to the cellular compensatory response, and revealed hundreds of differentially phosphorylated sites. Commonly downregulated phosphorylation sites were found on proteins involved in the cell cycle and RNA splicing. Our phosphoproteomics also suggested key regulators immediately responding to LAT1 inhibition. Focusing on one of these regulators, protein kinase CK2, we revealed LAT1 inhibition decreased phosphorylation of CK2 substrate without changing CK2 enzymatic activity. Furthermore, LAT1 inhibition abolished interaction between CK2 and its regulatory protein NOLC1, which suggests regulatory mechanism of CK2 substrate protein specificity controlled by LAT1 inhibition. Moreover, we revealed that the combination of JPH203 with CK2 inhibitor resulted in the enhanced inhibition of proliferation and migration of BTC cells.
CONCLUSION: This study provides new perspectives on LAT1-dependent cellular processes and a rationale for therapeutics targeting reprogrammed cancer metabolism.

Keywords:  Amino acid transporter system; Drug combinations; Neoplasms; Proteomics

DOI:  https://doi.org/10.1186/s40170-022-00295-8
Front Cell Dev Biol. 2022 ;10 1013001

Multiple congenital malformations arise from somatic mosaicism for constitutively active Pik3ca signaling.

Elise Marechal, Anne Poliard, Kilian Henry, Mathias Moreno, Mathilde Legrix, Nicolas Macagno, Grégoire Mondielli, Teddy Fauquier, Anne Barlier, Heather C Etchevers.

  Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.

Keywords:  PI3K; birth defect; cancer; cleft palate; embryo; neural crest; vascular anomaly

DOI:  https://doi.org/10.3389/fcell.2022.1013001
Biol Open. 2022 Sep 29. pii: bio.059544. [Epub ahead of print]

The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult mouse gut.

R Ramadan, V M Wouters, S M van Neerven, N E de Groot, Garcia T Martins, V Muncan, O D Franklin, M Battle, K S Carlson, J Leach, O J Sansom, O Boulard, M Chamaillard, L Vermeulen, J P Medema, D J Huels.

  The rapid of the epithelial gut lining is fueled by stem cells that reside at the base of intestinal crypts. The signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterized. In contrast, although extracellular matrix (ECM) components form an integral part of the intesti-nal stem cell niche, their direct influence on the cellular compo-sition is less well understood. We set out to systematically compare the effect of two ECM classes, interstitial matrix and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all cell types present in both cultures, albeit at different ratios. The collagen cultures contained a subset of cells enriched in fetal-like markers. In con-trast, laminin increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture, re-sembles the gut development in vivo. The ECM dramatic remodelling is accompa-nied by a local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. Importantly, deletion of laminin in the adult mouse results in a marked reduction of adult intestinal stem cells. Overall, our data support the hy-pothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.

Keywords:  Crypt morphology; Extracellular matrix; Intestinal stem cells; Laminin

DOI:  https://doi.org/10.1242/bio.059544
Cell Mol Gastroenterol Hepatol. 2022 Nov 07. pii: S2352-345X(22)00231-4. [Epub ahead of print]

Axin1 protects colon carcinogenesis by an immune-mediated effect.

Romain Sanson, Silvia Luna Lazzara, David Cune, Caterina Luana Pitasi, Coralie Trentesaux, Marie Fraudeau, Franck Letourneur, Benjamin Saintpierre, Morgane Le Gall, Pascale Bossard, Benoit Terris, Pascal Finetti, François Bertucci, Emilie Mamessier, Béatrice Romagnolo, Christine Perret.

   BACKGROUND & AIMS: Axin1 is a negative regulator of Wnt/β-catenin signaling with tumor suppressor function. The Wnt pathway has a critical role in the intestine, both during homeostasis and cancer, but the role of Axin1 remains elusive.
METHODS: We assessed the role of Axin1 in normal intestinal homeostasis, with control, epithelial-specific Axin1-knockout mice (Axin1ΔIEC) and Axin2-knockout mice. We evaluated the tumor suppressor function of Axin1 during chemically induced colorectal tumorigenesis and DSS-induced colitis, and performed comparative gene expression profiling by whole-genome RNA sequencing. The clinical relevance of the Axin1-dependent gene expression signature was then tested in a database of 2,239 clinical colorectal cancer (CRC) samples.
RESULTS: We found that Axin1 was dispensable for normal intestinal homeostasis and redundant with Axin2 for the Wnt pathway downregulation. Axin1 deficiency in intestinal epithelial cells rendered mice more susceptible to chemically induced colon carcinogenesis, but reduced DSS-induced colitis by attenuating the induction of a pro-inflammatory program. RNA-seq analyses identified an IFNγ/Th1 immune program controlled by Axin1 that enhances the inflammatory response and protects against colorectal cancer. The Axin1-dependent gene expression signature was applied to human CRC samples and identified a group of patients with potential vulnerability to immune checkpoint blockade therapies.
CONCLUSIONS: Our study establishes, in vivo, that Axin1 have redundant function with Axin2 for Wnt downregulation and infers a new role for Axin1. Physiologically, Axin1 stimulates gut inflammation via an IFNγ/Th1 program that prevent tumor growth. Linked to its T-cell mediated effect, the colonic Axin1 signature offers therapeutic perspectives for CRC.

Keywords:  Axin; Intestinal homeostasis; Wnt/β-catenin pathway; colon carcinogenesis; intestinal inflammation

DOI:  https://doi.org/10.1016/j.jcmgh.2022.10.017
Cancer Sci. 2022 Nov 12.

Rac1 promotes the reprogramming of glucose metabolism and the growth of colon cancer cells through up-regulating SOX9.

Jiaxin Liang, Qiang Liu, Longzheng Xia, Jinguan Lin, Linda Oyang, Shiming Tan, Qiu Peng, Xianjie Jiang, Xuemeng Xu, Nayiyuan Wu, Yanyan Tang, Min Su, Xia Luo, Yiqing Yang, Qianjin Liao, Yujuan Zhou.

  Metabolic reprogramming is the survival rule of tumor cells, and tumor cells can meet their high metabolic requirements by changing the energy metabolism mode. Metabolic reprogramming of tumor cells is an important biochemical basis of tumor malignant phenotypes. Ras-related C3 botulinum toxin substrate 1 (Rac1) is abnormally expressed in a variety of tumors and plays an important role in the proliferation, invasion and migration of tumor cells. However, the role of Rac1 in tumor metabolic reprogramming is still unclear. Herein, we revealed that Rac1 was highly expressed in colon cancer tissues and cell lines. Rac1 promotes the proliferation, migration and invasion of colon cancer cells by upregulating SOX9, which as a transcription factor can directly bind to the promoters of HK2 and G6PD genes and regulate their transcriptional activity. Rac1 upregulates the expression of SOX9 through the PI3K/AKT signaling pathway. Moreover, Rac1 can promote glycolysis and the activation of pentose phosphate pathway in colon cancer cells by mediating the axis of SOX9/HK2/G6PD. These findings reveal novel regulatory axes involving Rac1/SOX9/HK2/G6PD in the development and progression of colon cancer, providing novel promising therapeutic targets.

Keywords:  Rac1; SOX9; glycolysis; metabolic reprogramming; pentose phosphate pathway

DOI:  https://doi.org/10.1111/cas.15652
Leukemia. 2022 Nov 09.

mTOR inhibition amplifies the anti-lymphoma effect of PI3Kβ/δ blockage in diffuse large B-cell lymphoma.

Wendan Xu, Philipp Berning, Tabea Erdmann, Michael Grau, Nardjas Bettazová, Myroslav Zapukhlyak, Fabian Frontzek, Corinna Kosnopfel, Peter Lenz, Michael Grondine, Brandon Willis, James T Lynch, Pavel Klener, Stephan Hailfinger, Simon T Barry, Georg Lenz.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kβ/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kβ/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kβ/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kβ/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kβ/δ signaling and thus identifies a previously unappreciated role of the PI3Kβ isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kβ/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.

DOI: https://doi.org/10.1038/s41375-022-01749-0