bims-pimaco 2021-06-13 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–06–13
thirteen papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis.
Oncogenic BRAF, unrestrained by TGFβ-receptor signalling, drives right-sided colonic tumorigenesis.
Random survival forests identify pathways with polymorphisms predictive of survival in KRAS mutant and KRAS wild-type metastatic colorectal cancer patients.
A patent review of mTOR inhibitors for cancer therapy (2011-2020).
Drug sensitivity profile of minor KRAS mutations in colorectal cancer using mix culture assay: The effect of AMG-510, a novel KRAS G12C selective inhibitor, on colon cancer cells is markedly enhanced by the combined inhibition of MEK and BCL-XL.
RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis.
An interdomain hydrogen bond in the Rag GTPases maintains stable mTORC1 signaling in sensing amino acids.
Scaphium affine Ethanol Extract Induces Anoikis by Regulating the EGFR/Akt Pathway in HCT116 Colorectal Cancer Cells.
Comparative metabolomics and lipidomics study to evaluate the metabolic differences between 1st - and 2nd -generation mTOR inhibitors.
Mutational Landscape of PI3K-AKT-mTOR Pathway in Breast Cancer: Implications for Targeted Therapeutics.
mTOR pathway and DNA damage response: A therapeutic strategy in cancer therapy.
IGF-1R depletion sensitizes colon cancer cell lines to radiotherapy.
The Intestinal Epithelium - Fluid Fate and Rigid Structure From Crypt Bottom to Villus Tip.

Nat Commun. 2021 06 08. 12(1): 3444

PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis.

Shasha Yin, Liu Liu, Charles Brobbey, Viswanathan Palanisamy, Lauren E Ball, Shaun K Olsen, Michael C Ostrowski, Wenjian Gan.

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.

DOI: https://doi.org/10.1038/s41467-021-23833-2
Nat Commun. 2021 06 08. 12(1): 3464

Oncogenic BRAF, unrestrained by TGFβ-receptor signalling, drives right-sided colonic tumorigenesis.

S:CORT consortium

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFβ signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFβ-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFβ-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

DOI: https://doi.org/10.1038/s41467-021-23717-5
Sci Rep. 2021 Jun 09. 11(1): 12191

Random survival forests identify pathways with polymorphisms predictive of survival in KRAS mutant and KRAS wild-type metastatic colorectal cancer patients.

Madiha Naseem, Shu Cao, Dongyun Yang, Joshua Millstein, Alberto Puccini, Fotios Loupakis, Sebastian Stintzing, Chiara Cremolini, Ryuma Tokunaga, Francesca Battaglin, Shivani Soni, Martin D Berger, Afsaneh Barzi, Wu Zhang, Alfredo Falcone, Volker Heinemann, Heinz-Josef Lenz.

KRAS status serves as a predictive biomarker of response to treatment in metastatic colorectal cancer (mCRC). We hypothesize that complex interactions between multiple pathways contribute to prognostic differences between KRAS wild-type and KRAS mutant patients with mCRC, and aim to identify polymorphisms predictive of clinical outcomes in this subpopulation. Most pathway association studies are limited in assessing gene-gene interactions and are restricted to an individual pathway. In this study, we use a random survival forests (RSF) method for identifying predictive markers of overall survival (OS) and progression-free survival (PFS) in mCRC patients treated with FOLFIRI/bevacizumab. A total of 486 mCRC patients treated with FOLFIRI/bevacizumab from two randomized phase III trials, TRIBE and FIRE-3, were included in the current study. Two RSF approaches were used, namely variable importance and minimal depth. We discovered that Wnt/β-catenin and tumor associated macrophage pathway SNPs are strong predictors of OS and PFS in mCRC patients treated with FOLFIRI/bevacizumab independent of KRAS status, whereas a SNP in the sex-differentiation pathway gene, DMRT1, is strongly predictive of OS and PFS in KRAS mutant mCRC patients. Our results highlight RSF as a useful method for identifying predictive SNPs in multiple pathways.

DOI: https://doi.org/10.1038/s41598-021-91330-z
Expert Opin Ther Pat. 2021 Jun 08.

A patent review of mTOR inhibitors for cancer therapy (2011-2020).

Han-Yue Qiu, Peng-Fei Wang, Min Zhang.

   INTRODUCTION: The mammalian target of rapamycin (mTOR) kinase is a central component in the PI3K/Akt/mTOR pathway and plays a crucial role in tumor biology, making it one appealing therapeutic target. In the past decade, the mTORi (mTOR inhibitor) development field has made great progress, with more agents entering key trials and the proposal of third-generation mTORi concept. Yet to achieve significant clinical success, combined efforts from multiple disciplines are ever needed.
AREAS COVERED: This review focuses on the progress of mTORi development with anticancer potential from the perspective of the patent literature proposed between 2011 to 2020.
EXPERT OPINION: The highly complex regulatory mechanism network of mTOR proposes huge challenges to the development of clinically efficient mTORis. While in-depth biological research and fundamental medchemistry research are of importance to provide guidelines for improving mTORis, new technologies to pre-diagnose applicable populations is another key to provide precise personal cancer treatment. New mTOR agents are ever needed to tackle the common problems of side effects and drug resistance.

Keywords:  cancer treatment; mTOR inhibitors; mTOR/PI3K dualrapalink; rapalogsATP-competitive

DOI:  https://doi.org/10.1080/13543776.2021.1940137
Mol Clin Oncol. 2021 Jul;15(1): 148

Drug sensitivity profile of minor KRAS mutations in colorectal cancer using mix culture assay: The effect of AMG-510, a novel KRAS G12C selective inhibitor, on colon cancer cells is markedly enhanced by the combined inhibition of MEK and BCL-XL.

Masato Kitazawa, Yusuke Miyagawa, Makoto Koyama, Satoshi Nakamura, Nao Hondo, Satoru Miyazaki, Futoshi Muranaka, Shigeo Tokumaru, Yuta Yamamoto, Takehito Ehara, Masatsugu Kuroiwa, Hirokazu Tanaka, Daisuke Komatsu, Michiko Takeoka, Yuji Soejima.

  Colorectal cancer with a Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) gene mutation is considered to be resistant to anti-EGFR agents. G12D is the most common KRAS mutation in colorectal cancer, followed by G12V and G13D. According to clinical and basic research data, patients with colorectal cancer exhibiting G12D and G12V KRAS mutations are resistant to anti-EGFR agents; however, this is not true of G13D and other minor mutations, which are still not well understood. The current study focused on minor KRAS mutations (G12A, G12C, G12S, Q61H and A146T) and evaluated whether these were resistant to anti-EGFR antibodies using a mix culture assay. The results demonstrated that all KRAS mutations, including minor mutations, were resistant to two anti-EGFR agents: Cetuximab and panitumumab. The combined effect of MEK and BCL-XL inhibition on colorectal cancer cells with KRAS minor mutations were subsequently evaluated. The combined effect of MEK and BCL-XL inhibitors was confirmed in all KRAS minor mutations. The sensitivity of AMG510, a novel KRAS G12C selective inhibitor, was also assessed. The mix culture assay revealed that AMG510 selectively exerted an antitumor effect on colon cancer cells with a G12C KRAS mutation. The combination of MEK and BCL-XL inhibition markedly enhanced the effect of AMG510 in colon cancer cells. The current study suggested that AMG510 may have potential clinical use in combination with MEK and BCL-XL inhibitors in the treatment of patients with colorectal cancer exhibiting the G12C KRAS mutation.

Keywords:  ABT263; AMG510; BCL-XL; G12C; Kirsten rat sarcoma 2 viral oncogene homolog; MEK; colorectal cancer; trametinib

DOI:  https://doi.org/10.3892/mco.2021.2310
Elife. 2021 Jun 07. pii: e63807. [Epub ahead of print]10

RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis.

Máté Nászai, Karen Bellec, Yachuan Yu, Alvaro Román-Fernández, Emma Sandilands, Joel Johansson, Andrew D Campbell, Jim C Norman, Owen J Sansom, David M Bryant, Julia B Cordero.

  RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.

Keywords:  D. melanogaster; cancer biology; human; mouse; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.63807
J Biol Chem. 2021 Jun 08. pii: S0021-9258(21)00661-X. [Epub ahead of print] 100861

An interdomain hydrogen bond in the Rag GTPases maintains stable mTORC1 signaling in sensing amino acids.

Shawn B Egri, Kuang Shen.

  Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1 (mTORC1), which balances nutrient availability against the cell's anabolic needs. Central to the activity of mTORC1 is the RagA-RagC GTPase heterodimer, which under favorable conditions recruits the complex to the lysosomal surface to promote its activity. The RagA-RagC heterodimer has a unique architecture in that both subunits are active GTPases. To promote mTORC1 activity, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, while the opposite nucleotide loading configuration inhibits this signaling pathway. Despite its unique molecular architecture, how the Rag GTPase heterodimer maintains the oppositely loaded nucleotide state remains elusive. Here, we applied structure-function analysis approach to the crystal structures of the Rag GTPase heterodimer, and identified a key hydrogen bond that stabilizes the GDP-loaded state of the Rag GTPases. This hydrogen bond is mediated by the backbone carbonyl of Asn30 in the nucleotide binding domain (NBD) of RagA or Lys84 of RagC, and the hydroxyl group on the side chain of Thr210 in the C-terminal roadblock domain (CRD) of RagA or Ser266 of RagC, respectively. Eliminating this interdomain hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in a distorted response to amino acid signals. Our results reveal that this long-distance interdomain interaction within the Rag GTPase is required for the maintenance and regulation of the mTORC1 nutrient-sensing pathway.

Keywords:  Rag GTPase; amino acid; enzyme mechanism; hydrogen bond; mTOR complex 1 (mTORC1); nutrient sensing

DOI:  https://doi.org/10.1016/j.jbc.2021.100861
Front Oncol. 2021 ;11 621346

Scaphium affine Ethanol Extract Induces Anoikis by Regulating the EGFR/Akt Pathway in HCT116 Colorectal Cancer Cells.

Hye Won Kawk, Gun-He Nam, Myeong Jin Kim, Sang-Yong Kim, Young-Min Kim.

  Scaphium affine ethanol extracts (SAE) is a species that has been shown to contain various physiological effects; however, its anticancer effects have yet to be revealed. We qualitatively evaluated β-sitosterol in SAE through high-performance liquid chromatography (HPLC). The cytotoxicity in HCT116 and HT29 colorectal cancer cells and CCD841 normal colon cells was confirmed through WST-1 assays. Selective cytotoxicity was observed in colorectal cancer cells, with greater cytotoxicity demonstrated in the HCT116 cell line. As such, the HCT116 colorectal cell line was selected for subsequent experiments. After HCT116 cells were treated with SAE, it was confirmed that the apoptosis rate was increased in a SAE dose-dependent manner through Annexin V assay. SAE further showed dose-dependent suppression of invasion through invasion assays. Anoikis induction through the EGFR/Akt pathway in HCT116 colorectal cancer cells was confirmed by Western blotting. The tumor suppressive effects of SAE was assessed in vivo using a xenograft model of human HCT116 colorectal cancer cells. As a result, we confirmed that SAE decreased tumor size in a dose-dependent manner and that p-EGFR and cleaved-caspase 3 in tumors were also regulated in a dose-dependent manner. This study showed that SAE, by containing β-sitosterol with proven anticancer effects, induces anoikis through the EGFR/Akt pathway in HCT116 colorectal cancer cells both in vitro and in vivo.

Keywords:  EGFR/Akt pathway; HCT116 colorectal cancer cells; Scaphium affine; anoikis; xenograft model

DOI:  https://doi.org/10.3389/fonc.2021.621346
Biomed Chromatogr. 2021 Jun 08. e5190

Comparative metabolomics and lipidomics study to evaluate the metabolic differences between 1st - and 2nd -generation mTOR inhibitors.

Md Mamunur Rashid, Hyunbeom Lee, Jinyoung Park, Byung Hwa Jung.

  Mammalian target of rapamycin (mTOR) drives its fundamental cellular functions through two distinct catalytic subunits, mTORC1 and mTORC2, and is frequently dysregulated in most cancers. To treat cancers, developed mTOR inhibitors have been classified into 1st - and 2nd -generations based on their ability to inhibit single (1st -generation) and dual (2nd -generation) mTOR subunits. However, the underlying metabolic differences due to the effects of 1st - and 2nd -generation mTOR inhibitors have not been clearly evaluated. In this study, rapamycin (sirolimus), and AZD8055 and PP242 were selected as 1st - and 2nd -generation mTOR inhibitors, respectively, to evaluate the metabolic differences due to these two generations of mTOR inhibitors after a single oral dose using untargeted metabolomics and lipidomics approaches. The metabolic differences at each time point were compared using multivariate analysis. The multivariate and data analyses showed that metabolic disparity was more prominent within 8 h after drug administration and a broad class of metabolites was affected by the administration of both generations of mTOR inhibitors. Among the metabolite classes, changes in the pattern of fatty acids and glycerophospholipids were opposite, specifically at 4 and 8 h between the two generations mTOR inhibitors. We speculate that the inhibition of the mTORC2 subunit by the 2nd -generation mTOR inhibitor may have resulted in a distinct metabolic pattern between the 1st - and 2nd -generation inhibitors. Finally, the findings of this study could assist in a more detailed understanding of the key metabolic differences caused by 1st - and 2nd -generation mTOR inhibitors.

Keywords:  Lipidomics; Metabolomics; UHPLC-Orbitrap-MS; mTOR; mTOR inhibitor

DOI:  https://doi.org/10.1002/bmc.5190
J Cancer. 2021 ;12(14): 4408-4417

Mutational Landscape of PI3K-AKT-mTOR Pathway in Breast Cancer: Implications for Targeted Therapeutics.

Weikai Xiao, Guochun Zhang, Bo Chen, Xiaoqing Chen, Lingzhu Wen, Jianguo Lai, Xuerui Li, Min Li, Hao Liu, Jing Liu, Han Han-Zhang, Analyn Lizaso, Ning Liao.

  Background: Comprehensive analysis of PI3K-AKT-mTOR pathway gene alterations in breast cancer may be helpful for targeted therapy. Methods: We performed targeted sequencing using a panel of 520 cancer-related genes to investigate gene alterations in the PI3K-AKT-mTOR pathway from 589 consecutive Chinese women diagnosed with stage I-III breast cancer. Analyses of overall survival (OS) were performed using the publicly available clinical and genomic data from METABRIC. Results: PI3K-AKT-mTOR pathway gene alterations were detected in 62.6% (369/589) of our cohort. The most commonly altered genes were PIK3CA (45%), PTEN (7.5%), AKT1 (5.9 %), PIK3R1 (2.7%), and PIK3CG (2%). Four PIK3CA mutations (E545K, H1047R, E542K, and H1047L) were detected in all the breast cancer molecular subtypes. Seven PIK3CA mutations (E545G, E418_L422delinsV, E726K, E110del, G1049R, G118D, and D350G) were only detected in HR+ subtypes. Two PIK3CA mutations (C420R and N345K) were only detected in non-triple-negative subtypes. Most cases with PTEN mutation were HR+/HER2- subtype (77.3%), followed by triple-negative subtype (18.2%). In the METABRIC breast cancer dataset, no significant OS difference was observed between the PIK3CA-mutant and wild-type groups. However, patients with multiple PIK3CA mutations (mOS: 131 vs. 159 months, P= 0.029), or PIK3CA mutations located in the C2 domain had significantly shorter OS (mOS, 130 vs. 154 months, P=0.020) than those without the mutations. Conclusions: Our study reveals the heterogeneity in PI3K-AKT-mTOR pathway among the breast cancer molecular subtypes in our cohort. Moreover, the number and specific sites of PIK3CA mutations have distinct prognostic impact.

Keywords:  Breast cancer; PI3K-AKT-mTOR pathway; gene alteration; molecular subtypes; prognosis

DOI:  https://doi.org/10.7150/jca.52993
DNA Repair (Amst). 2021 Jun 03. pii: S1568-7864(21)00098-7. [Epub ahead of print]104 103142

mTOR pathway and DNA damage response: A therapeutic strategy in cancer therapy.

Romina Danesh Pazhooh, Parnia Rahnamay Farnood, Zatollah Asemi, Liaosadat Mirsafaei, Bahman Yousefi, Hamed Mirzaei.

  The mammalian target of rapamycin (mTOR) is a conserved serine/threonine-protein kinase, comprising two subunit protein complexes: mTORC1 and mTORC2. In response to insult and cancer, the mTOR pathway plays a crucial role in regulating growth, metabolism, cell survival, and protein synthesis. Key subunits of mTORC1/2 catalyze the phosphorylation of various molecules, including eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), ribosomal protein S6 kinase β-1 (S6K1). The DNA damage response (DDR) maintains genomic stability and provides an opportunity for treating tumors with defects caused by DNA damaging agents. Many mTOR inhibitors are utilized for the treatment of cancers. However, several clinical trials are still assessing the efficacy of mTOR inhibitors. This paper discusses the role of the mTOR signaling pathway and its regulators in developing cancer. In the following, we will review the interaction between DDR and mTOR signaling and the innovative therapies applied in preclinical and clinical trials for treating cancers.

Keywords:  Cancer; DNA damage; DNA repair; Therapy; mTOR

DOI:  https://doi.org/10.1016/j.dnarep.2021.103142
Cancer Biomark. 2021 Jun 05.

IGF-1R depletion sensitizes colon cancer cell lines to radiotherapy.

Rui Zong, Xiaoman Chen, Jingjing Feng, Shan Xu.

   PURPOSE: Insulin like growth factor receptor 1 (IGF-1R) has been documented to play a key role in radiation response, thereby offering an attractive drug target to enhance tumor sensitivity to radiotherapy. Here, we investigated weather knockdown of IGF-1R can sensitize colorectal cancer (CRC) cell lines to radiation.
MATERIAL AND METHODS: Human colon carcinoma SW480 and HT-29 cells were transfected with specific small interference RNA (siRNA) to mediate IGF-1R depletion. The expression of IGF-1R mRNA and protein among transfected and untransfected cells was detected by Western blot analysis. Changes in cell proliferation and radiosensitivity were evaluated by the clonogenic survival assay. NVP-ADW742, an IGF-1R inhibitor, in combination with radiation was studied. RAD51, a measure for homologous recombination repair, and 53BP1, a maker for non-homologous end-joining (NHEJ), were determined by immunofluorescence for double-strand breaks (DSB) repair pathways. Cell cycle was also examined in the IGF-1R knockdown and IGF-1R-inhibited cells.
RESULTS: CRC cell lines were selectively sensitized to radiation after siRNA-mediated IGF-1R depletion. NVP-ADW742 efficiently increases cancer cell response to radiation. Furthermore, initial formation of RAD51 foci after IR, and 53BP1 foci were significantly reduced in IGF-1R-depleted or with IGF-1R Inhibitor CRC cell lines. Lastly, IGF-1R-depleted or with IGF-1R Inhibitor caused more G2 phase cell arrest.
CONCLUSION: Our findings demonstrate that depletion of IGF-1R lead to an increase in radiosensitivity in CRC.

Keywords:  Colorectal cancer; IGF-1R; neoadjuvant radiotherapy; radiosensitivity

DOI:  https://doi.org/10.3233/CBM-210016
Front Cell Dev Biol. 2021 ;9 661931

The Intestinal Epithelium - Fluid Fate and Rigid Structure From Crypt Bottom to Villus Tip.

Vangelis Bonis, Carla Rossell, Helmuth Gehart.

  The single-layered, simple epithelium of the gastro-intestinal tract controls nutrient uptake, coordinates our metabolism and shields us from pathogens. Despite its seemingly simple architecture, the intestinal lining consists of highly distinct cell populations that are continuously renewed by the same stem cell population. The need to maintain balanced diversity of cell types in an unceasingly regenerating tissue demands intricate mechanisms of spatial or temporal cell fate control. Recent advances in single-cell sequencing, spatio-temporal profiling and organoid technology have shed new light on the intricate micro-structure of the intestinal epithelium and on the mechanisms that maintain it. This led to the discovery of unexpected plasticity, zonation along the crypt-villus axis and new mechanism of self-organization. However, not only the epithelium, but also the underlying mesenchyme is distinctly structured. Several new studies have explored the intestinal stroma with single cell resolution and unveiled important interactions with the epithelium that are crucial for intestinal function and regeneration. In this review, we will discuss these recent findings and highlight the technologies that lead to their discovery. We will examine strengths and limitations of each approach and consider the wider impact of these results on our understanding of the intestine in health and disease.

Keywords:  cancer; differentiation; intestine; organoid; plasticity; regeneration; single cell; stem cell

DOI:  https://doi.org/10.3389/fcell.2021.661931