bims-pimaco 2020-12-06 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2020–12–06
nine papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus.
AZD0364 is a potent and selective ERK1/2 inhibitor which enhances anti-tumour activity in KRAS mutant tumour models when combined with the MEK inhibitor selumetinib.
NRF2 and the Ambiguous Consequences of Its Activation during Initiation and the Subsequent Stages of Tumourigenesis.
Serial Circulating Tumor DNA Mutational Status in Patients with KRAS-Mutant Metastatic Colorectal Cancer from the Phase 3 AIO KRK0207 Trial.
Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.
The Role of BRAF in Metastatic Colorectal Carcinoma-Past, Present, and Future.
Stomach-specific c-Myc overexpression drives gastric adenoma in mice via AKT/mTOR signaling.
RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.
Influence of Cholesterol on the Orientation of the Farnesylated GTP-Bound KRas-4B Binding with Anionic Model Membranes.

Nat Commun. 2020 11 30. 11(1): 6088

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus.

Xin Zhou, Yanghao Zhong, Olivia Molinar-Inglis, Maya T Kunkel, Mingyuan Chen, Tengqian Sun, Jiao Zhang, John Y-J Shyy, JoAnn Trejo, Alexandra C Newton, Jin Zhang.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.

DOI: https://doi.org/10.1038/s41467-020-19937-w
Mol Cancer Ther. 2020 Dec 03. pii: molcanther.0002.2020. [Epub ahead of print]

AZD0364 is a potent and selective ERK1/2 inhibitor which enhances anti-tumour activity in KRAS mutant tumour models when combined with the MEK inhibitor selumetinib.

Vikki Flemington, Emma J Davies, David Robinson, Linda C Sandin, Oona Delpuech, Pei Zhang, Lyndsey Hanson, Paul Farrington, Sigourney Bell, Katarzyna Falenta, Francis D Gibbons, Nicola Lindsay, Aaron Smith, Joanne Wilson, Karen Roberts, Michael Tonge, Philip Hopcroft, Sophie E Willis, Martine P Roudier, Claire Rooney, Elizabeth A Coker, Patricia Jaaks, Mathew J Garnett, Stephen E Fawell, Clifford D Jones, Richard A Ward, Iain Simpson, Sabina C Cosulich, J Elizabeth Pease, Paul D Smith.

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signalling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF mutant melanoma, with limited effect of single agent pathway inhibitors in KRAS mutant tumours. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signalling in KRAS mutant tumours and achieve meaningful clinical benefit. Here we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF mutant and KRAS mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose and time-dependent modulation of ERK1/2-dependent signalling biomarkers resulting in tumour regression in sensitive BRAF and KRAS mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor selumetinib (AZD6244, ARRY142886) enhances efficacy in KRAS mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signalling pathway that is not achievable with single agent treatment. The AZD0364 and selumetinib combination also results in significant tumour regressions in multiple KRAS mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS mutant tumours.

DOI: https://doi.org/10.1158/1535-7163.MCT-20-0002
Cancers (Basel). 2020 Dec 02. pii: E3609. [Epub ahead of print]12(12):

NRF2 and the Ambiguous Consequences of Its Activation during Initiation and the Subsequent Stages of Tumourigenesis.

Holly Robertson, Albena T Dinkova-Kostova, John D Hayes.

  NF-E2 p45-related factor 2 (NRF2, encoded in the human by NFE2L2) mediates short-term adaptation to thiol-reactive stressors. In normal cells, activation of NRF2 by a thiol-reactive stressor helps prevent, for a limited period of time, the initiation of cancer by chemical carcinogens through induction of genes encoding drug-metabolising enzymes. However, in many tumour types, NRF2 is permanently upregulated. In such cases, its overexpressed target genes support the promotion and progression of cancer by suppressing oxidative stress, because they constitutively increase the capacity to scavenge reactive oxygen species (ROS), and they support cell proliferation by increasing ribonucleotide synthesis, serine biosynthesis and autophagy. Herein, we describe cancer chemoprevention and the discovery of the essential role played by NRF2 in orchestrating protection against chemical carcinogenesis. We similarly describe the discoveries of somatic mutations in NFE2L2 and the gene encoding the principal NRF2 repressor, Kelch-like ECH-associated protein 1 (KEAP1) along with that encoding a component of the E3 ubiquitin-ligase complex Cullin 3 (CUL3), which result in permanent activation of NRF2, and the recognition that such mutations occur frequently in many types of cancer. Notably, mutations in NFE2L2, KEAP1 and CUL3 that cause persistent upregulation of NRF2 often co-exist with mutations that activate KRAS and the PI3K-PKB/Akt pathway, suggesting NRF2 supports growth of tumours in which KRAS or PKB/Akt are hyperactive. Besides somatic mutations, NRF2 activation in human tumours can occur by other means, such as alternative splicing that results in a NRF2 protein which lacks the KEAP1-binding domain or overexpression of other KEAP1-binding partners that compete with NRF2. Lastly, as NRF2 upregulation is associated with resistance to cancer chemotherapy and radiotherapy, we describe strategies that might be employed to suppress growth and overcome drug resistance in tumours with overactive NRF2.

Keywords:  ATF4; Cullin 3; KEAP1; NADPH generation; NRF2; adaptation; antioxidant; autophagy; bioactivation; bladder; chemoprevention; chemotherapy; colon; drug metabolism; drug resistance; glutathione; head and neck; initiation; liver; lung; metastasis; oesophagus; oncogene; oxidative stress; pentose phosphate pathway; progression; proteasome; quinone-containing drugs; reactive oxygen species; rectum; recurrent disease; stomach; thioredoxin; tumour suppressor

DOI:  https://doi.org/10.3390/cancers12123609
Clin Chem. 2020 Nov 30. pii: hvaa223. [Epub ahead of print]

Serial Circulating Tumor DNA Mutational Status in Patients with KRAS-Mutant Metastatic Colorectal Cancer from the Phase 3 AIO KRK0207 Trial.

Smiths S Lueong, Andreas Herbst, Sven-Thorsten Liffers, Nicola Bielefeld, Peter A Horn, Andrea Tannapfel, Anke Reinacher-Schick, Axel Hinke, Susanna Hegewisch-Becker, Frank T Kolligs, Jens T Siveke.

   BACKGROUND: We assessed the usefulness of circulating tumor DNA (ctDNA) pre- or post-treatment initiation for outcome prediction and treatment monitoring in metastatic colorectal cancer (mCRC).
METHODS: Droplet digital PCR was used to measure absolute mutant V-Ki-ras2 Kirsten rat sarcoma viral oncogene ((mut)KRAS) ctDNA concentrations in 214 healthy controls (plasma and sera) and in 151 tissue-based mutKRAS positive patients with mCRC from the prospective multicenter phase 3 trial AIO KRK0207. Serial mutKRAS ctDNA was analyzed prior to and 2-3 weeks after first-line chemotherapy initiation with fluoropyrimidine, oxaliplatin, and bevacizumab in patients with mCRC and correlated with clinical parameters.
RESULTS: mut KRAS ctDNA was detected in 74.8% (113/151) of patients at baseline and in 59.6% (90/151) at follow-up. mutKRAS ctDNA at baseline and follow-up was associated with poor overall survival (OS) (hazard ratio [HR] =1.88, 95% confidence interval [CI] 1.20-2.95; HR = 2.15, 95% CI 1.47-3.15) and progression-free survival (PFS) (HR = 2.53, 95% CI 1.44-4.46; HR = 1.90, 95% CI 1.23-2.95), respectively. mutKRAS ctDNA clearance at follow-up conferred better disease control (P = 0.0075), better OS (log-rank P = 0.0018), and PFS (log-rank P = 0.0018). Measurable positive mutKRAS ctDNA at follow-up was the strongest and most significant independent prognostic factor on OS in multivariable analysis (HR = 2.31, 95% CI 1.40-3.25).
CONCLUSIONS: Serial analysis of circulating mutKRAS concentrations in mCRC has prognostic value. Post treatment mutKRAS concentrations 2 weeks after treatment initiation were associated with therapeutic response in multivariable analysis and may be an early response predictor in patients receiving first-line combination chemotherapy.
CLINICALTRIALSGOV IDENTIFIER: NCT00973609.

Keywords:  ctDNA; ddPCR; liquid biopsy; metastatic colorectal cancer; mutant KRAS

DOI:  https://doi.org/10.1093/clinchem/hvaa223
Proc Natl Acad Sci U S A. 2020 Nov 30. pii: 202005712. [Epub ahead of print]

Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.

Mo Zhou, Leena Kuruvilla, Xiarong Shi, Stephen Viviano, Ian M Ahearn, Caroline R Amendola, Wenjuan Su, Sana Badri, James Mahaffey, Nicole Fehrenbacher, Jane Skok, Joseph Schlessinger, Benjamin E Turk, David A Calderwood, Mark R Philips.

  Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

Keywords:  KRAS; RAS; SAFB; farnesyltransferase; prenyltransferase

DOI:  https://doi.org/10.1073/pnas.2005712117
Int J Mol Sci. 2020 Nov 26. pii: E9001. [Epub ahead of print]21(23):

The Role of BRAF in Metastatic Colorectal Carcinoma-Past, Present, and Future.

Angela Djanani, Silvia Eller, Dietmar Öfner, Jakob Troppmair, Manuel Maglione.

  With a global incidence of 1.8 million cases, colorectal cancer represents one of the most common cancers worldwide. Despite impressive improvements in treatment efficacy through cytotoxic and biological agents, the cancer-related death burden of metastatic colorectal cancer (mCRC) is still high. mCRC is not a genetically homogenous disease and various mutations influence disease development. Up to 12% of mCRC patients harbor mutations of the signal transduction molecule BRAF, the most prominent being BRAFV600E. In mCRC, BRAFV600E mutation is a well-known negative prognostic factor, and is associated with a dismal prognosis. The currently approved treatments for BRAF-mutated mCRC patients are of little impact, and there is no treatment option superior to others. However, the gradual molecular understanding over the last decades of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway, resulted in the development of new therapeutic strategies targeting the involved molecules. Recently published and ongoing studies administering a combination of different inhibitors (e.g., BRAF, MEK, and EGFR) showed promising results and represent the new standard of care. In this review, we present, both, the molecular and clinical aspects of BRAF-mutated mCRC patients, and provide an update on the current and future treatment approaches that might direct the therapy of mCRC in a new era.

Keywords:  BRAF; BRAF inhibitors; MAPK; colorectal cancer; multitargeted therapy

DOI:  https://doi.org/10.3390/ijms21239001
Bosn J Basic Med Sci. 2020 Nov 19.

Stomach-specific c-Myc overexpression drives gastric adenoma in mice via AKT/mTOR signaling.

Jing Liu, Wenxin Feng, Min Liu, Hanyu Rao, Xiaoxue Li, Yan Teng, Xiao Yang, Jin Xu, Weiqiang Gao, Li Li.

Gastric cancer (GC) is one of the most common malignant cancers in the world. c-Myc, a well-known oncogene, is commonly amplified in many cancers, including gastric cancer. However, it is still not completely understood how c-Myc functions in GC. Here, we generated a stomach-specific c-Myc transgenic mouse model to investigate its role in GC. We found that overexpression of c-Myc in Atp4b+ gastric parietal cells could induce gastric adenoma in mice. Mechanistically, c-Myc promoted tumorigenesis via the AKT/mTOR pathway. Furthermore, AKT inhibitor (MK-2206) or mTOR inhibitor (Rapamycin) inhibited the proliferation of c-Myc overexpressing gastric cancer cell lines. Thus, our findings highlight that gastric tumorigenesis can be induced by c-Myc overexpression through activation of the AKT/mTOR pathway.

DOI: https://doi.org/10.17305/bjbms.2020.4978
Mol Cell. 2020 Nov 23. pii: S1097-2765(20)30786-3. [Epub ahead of print]

RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.

Ayaz Najafov, Hoang Son Luu, Adnan K Mookhtiar, Lauren Mifflin, Hong-Guang Xia, Palak P Amin, Alban Ordureau, Huibing Wang, Junying Yuan.

  The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.

Keywords:  AMPK; CASP8; MLKL; RIPK1; RIPK3; TSC2; lysosome; mTORC1; neonatal lethality

DOI:  https://doi.org/10.1016/j.molcel.2020.11.008
Membranes (Basel). 2020 Nov 22. pii: E364. [Epub ahead of print]10(11):

Influence of Cholesterol on the Orientation of the Farnesylated GTP-Bound KRas-4B Binding with Anionic Model Membranes.

Huixia Lu, Jordi Martí.

  The Ras family of proteins is tethered to the inner leaflet of the cell membranes which plays an essential role in signal transduction pathways that promote cellular proliferation, survival, growth, and differentiation. KRas-4B, the most mutated Ras isoform in different cancers, has been under extensive study for more than two decades. Here we have focused our interest on the influence of cholesterol on the orientations that KRas-4B adopts with respect to the plane of the anionic model membranes. How cholesterol in the bilayer might modulate preferences for specific orientation states is far from clear. Herein, after analyzing data from in total 4000 ns-long molecular dynamics (MD) simulations for four KRas-4B systems, properties such as the area per lipid and thickness of the membrane as well as selected radial distribution functions, penetration of different moieties of KRas-4B, and internal conformational fluctuations of flexible moieties in KRas-4B have been calculated. It has been shown that high cholesterol content in the plasma membrane (PM) favors one orientation state (OS1), exposing the effector-binding loop for signal transduction in the cell from the atomic level. We confirm that high cholesterol in the PM helps KRas-4B mutant stay in its constitutively active state, which suggests that high cholesterol intake can increase mortality and may promote cancer progression for cancer patients. We propose that during the treatment of KRas-4B-related cancers, reducing the cholesterol level in the PM and sustaining cancer progression by controlling the plasma cholesterol intake might be taken into account in anti-cancer therapies.

Keywords:  HVR; KRas-4B; anionic plasma membrane; cholesterol; mutation; post-translational modification; signaling

DOI:  https://doi.org/10.3390/membranes10110364