bims-pimaco 2021-10-03 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–10–03
seven papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Cancer Signaling Drives Cancer Metabolism: AKT and the Warburg Effect.
Plasma ctDNA RAS status selects patients for anti-EGFR treatment rechallenge in metastatic colorectal cancer: a meta-analysis.
NRF2 Mediates Therapeutic Resistance to Chemoradiation in Colorectal Cancer through a Metabolic Switch.
MYC hyperactivates WNT signaling in APC/CTNNB1-mutated colorectal cancer cells through miR-92a-dependent repression of DKK3.
Suppression of colon tumorigenesis in mutant Apc mice by a novel PDE10 inhibitor that reduces oncogenic β-catenin.
Negative regulation of TGFβ-induced apoptosis by RAC1B enhances intestinal tumourigenesis.
A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

Cancer Res. 2021 Oct 01. 81(19): 4896-4898

Cancer Signaling Drives Cancer Metabolism: AKT and the Warburg Effect.

Aaron M Hosios, Brendan D Manning.

The Warburg effect, the propensity of some cells to metabolize glucose to lactate in the presence of oxygen (also known as aerobic glycolysis), has long been observed in cancer and other contexts of cell proliferation, but only in the past two decades have significant gains been made in understanding how and why this metabolic transformation occurs. In 2004, Cancer Research published a study by Elstrom and colleagues that provided one of the first connections between a specific oncogene and aerobic glycolysis. Studying hematopoietic and glioblastoma cell lines, they demonstrated that constitutive activation of AKT promotes an increased glycolytic rate without altering proliferation or oxygen consumption in culture. They proposed that it is this effect that allows constitutive AKT activation to transform cells and found that it sensitizes cells to glucose deprivation. In the years since, mechanistic understanding of oncogenic control of metabolism, and glycolysis specifically, has deepened substantially. Current work seeks to understand the benefits and liabilities associated with glycolytic metabolism and to identify inhibitors that might be of clinical benefit to target glycolytic cancer cells.See related article by Elstrom and colleagues, Cancer Res 2004;64:3892-9.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2647
Exp Oncol. 2021 09;43(3): 252-256

Plasma ctDNA RAS status selects patients for anti-EGFR treatment rechallenge in metastatic colorectal cancer: a meta-analysis.

M S Vlachou, D Mauri, G Zarkavelis, P Ntellas, C Tagkas, S Gkoura, G Pentheroudakis.

BACKGROUND: Recently one randomized trial and several phase II studies underscored that patients with metastatic colorectal cancer who progressed after an initial clinical benefit from anti-epidermal growth factor receptor (EGFR) treatment may further benefit from rechallenge with anti-EGFR therapy. Testing circulating tumor DNA (ctDNA) RAS status prior to anti-EGFR rechallenge seems a promising non-invasive method to predict and monitor response to anti-EGFR readministration.
AIM: To assess the capability of liquid biopsy ctDNA in exploring RAS status and in predicting outcome of metastatic colorectal cancer patients treated with anti-EGFR monoclonal antibody rechallenge.
MATERIALS AND METHODS: Systematic review of literature and meta-analysis of the available evidence.
RESULTS: Data from four studies involving 117 patients were available. All patients harbored RAS wild type tumors and derived benefit from first line anti-EGFR therapy. Of these, 65 underwent plasma ctDNA before anti-EGFR treatment rechallenge and were eligible for analyses: 35 patients had RAS wild type ctDNA, and 30 RAS mutated, indicating that 46% of patients underwent RAS status conversion after primary anti-EGFR therapy. Anti-EGFR rechallenge among patients with plasma ctDNA RAS wild type status was associated with a consistent benefit in progression free survival (hazard ratio (HR) 0.40, 95% confidence interval (CI) 0.22-0.70; p = 0.001; I2 = 0) and overall survival (HR 0.37, 95% CI 0.16-0.85; p = 0.02; I2 = 74%) when compared to its use among patients with plasma ctDNA RAS mutation. Patients with plasma ctDNA RAS wild type profile also performed statistically better in term of disease control rate, risk for disease progression at 3 and 6 months, and risk for death at 6 and 12 months.
CONCLUSION: RAS status assessment continues to be useful in predicting benefit for anti-EGFR treatment.
Antioxidants (Basel). 2021 Aug 28. pii: 1380. [Epub ahead of print]10(9):

NRF2 Mediates Therapeutic Resistance to Chemoradiation in Colorectal Cancer through a Metabolic Switch.

Séan M O'Cathail, Chieh-Hsi Wu, Rachael Thomas, Maria A Hawkins, Tim S Maughan, Annabelle Lewis.

  Radiation resistance is a significant clinical problem in rectal cancer treatment, the mechanisms of which are poorly understood. NRF2 signalling is known to contribute to chemo/radioresistance in some cancers, but its role in therapeutic resistance in colorectal cancer (CRC) is unexplored. Using siRNA and CRiSPR/Cas9 isogenic CRC cell lines, we investigated the effect of the knockdown and upregulation of the NRF2 pathway on chemo-radiosensitivity. Poly (A) enriched RNA sequencing and geneset enrichment analysis (GSEA) were carried out on both sensitive and resistant cell models for mechanistic insights. Finally, a cohort of rectal patient samples was profiled to understand the clinical relevance of NRF2 signalling. Radioresistant cell lines were significantly radiosensitised by siRNA knockdown (SW1463, SER10 1.22, ANOVA p < 0.0001; HT55, SER10 1.17, ANOVA p < 0.01), but not the (already) radiosensitive HCT116. The constitutive activation of NRF2 via a CRISPR Cas9 NFE2L2 mutation, E79K, induced radioresistance in HCT116 (SER10 0.71, ANOVA, p < 0.0001). GSEA demonstrated significant opposing metabolic dependencies in NRF2 signalling, specifically, the downregulation of amino acid and protein synthesis with low levels of NRF2 and upregulation with over expression. In a clinical cohort of 127 rectal patients, using a validated mRNA signature, higher baseline NRF2 signalling was associated with incomplete responses to radiation higher final neoadjuvant rectal (NAR) score (OR 1.34, 95% C.I. 1.01-1.80, LRT p-value = 0.023), where high NAR indicates poor radiation response and poor long-term prognosis. This is the first demonstration of NRF2-mediated radiation resistance in colorectal cancer. NRF2 appears to regulate crucial metabolic pathways, which could be exploited for therapeutic interventions.

Keywords:  NRF2; metabolism; radiation; therapeutic resistance

DOI:  https://doi.org/10.3390/antiox10091380
Mol Cancer Res. 2021 Sep 30. pii: molcanres.MCR-21-0666-A.2021. [Epub ahead of print]

MYC hyperactivates WNT signaling in APC/CTNNB1-mutated colorectal cancer cells through miR-92a-dependent repression of DKK3.

Priyanka Sehgal, Claudia Lanauze, Xin Wang, Katharina E Hayer, Manuel Torres-Diz, N Adrian Leu, Yogev Sela, Ben Z Stanger, Christopher J Lengner, Andrei Thomas-Tikhonenko.

Activation of Wnt signaling is among the earliest events of colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding β-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations in APC, a major β-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct β-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that Myc suppresses Dkk3 transcription through one of mir-17-92 cluster microRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in APC-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration and invasion and metastasis in a syngeneic orthotopic mouse colon cancer model. Implications: Our results suggest that the MYC->miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feedforward oncogenic loop.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0666
Cancer Prev Res (Phila). 2021 Sep 28. pii: canprevres.0208.2021. [Epub ahead of print]

Suppression of colon tumorigenesis in mutant Apc mice by a novel PDE10 inhibitor that reduces oncogenic β-catenin.

Kevin J Lee, Wen-Chi L Chang, Xi Chen, Jacob Valiyaveettil, Veronica Ramirez-Alcantara, Elaine Gavin, Alla Musiyenko, Luciana Madeira da Silva, Naga S Annamdevula, Silas J Leavesley, Antonio Ward, Tyler E Mattox, Ashley S Lindsey, Joel Andrews, Bing Zhu, Charles Wood, Ashleigh Neese, Ashley Nguyen, Kristy Berry, Yulia Y Maxuitenko, Mary Pat Moyer, Elmar Nurmemmedov, Greg Gorman, Lori Coward, Gang Zhou, Adam B Keeton, Harry S Cooper, Margie L Clapper, Gary A Piazza.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-cyclooxygenase inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer (CRC) without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing CRC.

DOI: https://doi.org/10.1158/1940-6207.CAPR-21-0208
Cell Death Dis. 2021 Sep 25. 12(10): 873

Negative regulation of TGFβ-induced apoptosis by RAC1B enhances intestinal tumourigenesis.

Victoria Gudiño, Patrizia Cammareri, Caroline V Billard, Kevin B Myant.

RAC1B is a tumour-related alternative splice isoform of the small GTPase RAC1, found overexpressed in a large number of tumour types. Building evidence suggests it promotes tumour progression but compelling in vivo evidence, demonstrating a role in driving tumour invasion, is currently lacking. In the present study, we have overexpressed RAC1B in a colorectal cancer mouse model with potential invasive properties. Interestingly, RAC1B overexpression did not trigger tumour invasion, rather it led to an acceleration of tumour initiation and reduced mouse survival. By modelling early stages of adenoma initiation we observed a reduced apoptotic rate in RAC1B overexpressing tumours, suggesting protection from apoptosis as a mediator of this phenotype. RAC1B overexpressing tumours displayed attenuated TGFβ signalling and functional analysis in ex vivo organoid cultures demonstrated that RAC1B negatively modulates TGFβ signalling and confers resistance to TGFβ-driven cell death. This work defines a novel mechanism by which early adenoma cells can overcome the cytostatic and cytotoxic effects of TGFβ signalling and characterises a new oncogenic function of RAC1B in vivo.

DOI: https://doi.org/10.1038/s41419-021-04177-7
Analyst. 2021 Sep 29.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

Bjoern C Froehlich, Robert Popp, Constance A Sobsey, Sahar Ibrahim, Andre LeBlanc, Yassene Mohammed, Marguerite Buchanan, Adriana Aguilar-Mahecha, Oliver Pötz, Michael X Chen, Alan Spatz, Mark Basik, Gerald Batist, René P Zahedi, Christoph H Borchers.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 μg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

DOI: https://doi.org/10.1039/d1an00165e