bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2021‒03‒28
fourteen papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research

  1. Ther Adv Med Oncol. 2021 ;13 1758835921992974
      Introduction: BRAF driver mutations are found in up to 15% of patients with colorectal cancer (CRC) and lead to constitutive activation of BRAF kinase and sustained RAS/RAF/MEK/ERK pathway signaling. BRAF mutations define a sub-population characterized by a poor prognosis and dismal median survival. Following successful outcomes with BRAF inhibition in BRAF mutant metastatic melanoma, this approach was evaluated in metastatic colorectal cancer (mCRC). The development and combination of targeted therapies against multiple signaling pathways has proved particularly successful, with improved survival and response rates.Areas covered: This review addresses the development of therapeutic strategies with inhibitors targeting MAPK/ERK and EGFR signaling in BRAF V600E mutated mCRC, focusing on encorafenib, binimetinib and cetuximab. A pharmacological and clinical review of these drugs and the therapeutic approaches behind their optimization are presented.
    Expert opinion: Exploiting knowledge of the mechanisms of resistance to BRAF inhibitors has been crucial to developing effective therapeutic strategies in BRAF-V600E mutant mCRC. The BEACON trial is a successful example of this approach, using encorafenib and cetuximab with or without binimetinib in patients with previously treated BRAF V600E mutant mCRC, showing an impressive improvement in clinical outcomes and tolerable toxicity compared with chemotherapy, establishing a new standard of care in this setting.
    Keywords:  BEACON clinical trial; BRAF V600E mutation; BRAF inhibitor; EGFR inhibitor; MEK inhibitor; binimetinib; colon cancer; encorafenib
  2. Biochem Biophys Res Commun. 2021 Mar 17. pii: S0006-291X(21)00427-7. [Epub ahead of print]552 106-113
      Cancer is characterized by uncontrolled proliferation resulting from aberrant cell cycle progression. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling, a regulatory pathway for the cell cycle, stabilizes cyclin D1 in the G1 phase by inhibiting the activity of glycogen synthase kinase 3β (GSK3β) via phosphorylation. We previously reported that phospholipase C-related catalytically inactive protein (PRIP), a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding protein, regulates PI3K/AKT signaling by competitively inhibiting substrate recognition by PI3K. Therefore, in this study, we investigated whether PRIP is involved in cell cycle progression. PRIP silencing in MCF-7 cells, a human breast cancer cell line, demonstrated PI(3,4,5)P3 signals accumulated at the cell periphery compared to that of the control. This suggests that PRIP reduction enhances PI(3,4,5)P3-mediated signaling. Consistently, PRIP silencing in MCF-7 cells exhibited increased phosphorylation of AKT and GSK3β which resulted in cyclin D1 accumulation. In contrast, the exogenous expression of PRIP in MCF-7 cells evidenced stronger downregulation of AKT and GSK3β phosphorylation, reduced accumulation of cyclin D1, and diminished cell proliferation in comparison to control cells. Flow cytometry analysis indicated that MCF-7 cells stably expressing PRIP attenuate cell cycle progression. Importantly, tumor growth of MCF-7 cells stably expressing PRIP was considerably prevented in an in vivo xenograft mouse model. In conclusion, PRIP expression downregulates PI3K/AKT/GSK3β-mediated cell cycle progression and suppresses tumor growth. Therefore, we propose that PRIP is a new therapeutic target for anticancer therapy.
    Keywords:  AKT; PI(3,4,5)P(3); PRIP; Phosphatidylinositol 3-kinase; Tumor growth
  3. Theranostics. 2021 ;11(9): 4335-4350
      Background: Cancer is a leading cause of death worldwide. Extensive research over decades has led to the development of therapies that inhibit oncogenic signaling pathways. The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in the development of many cancers. Several mTOR inhibitors are approved for the treatment of cancers. However, the anticancer efficacies of mTOR inhibitor monotherapy are still limited. Methods: Western blot was used to detect the expression of indicated molecules. Thioredoxin reductase (TrxR) activity in cells was determined by the endpoint insulin reduction assay. Immunofluorescence staining was used to analyze precise location and expression of target proteins. Nude mice were used for xenograft tumor models. Results: We identified a synergistic lethal interaction of mTOR and TrxR inhibitors and elucidated the underlying molecular mechanisms of this synergism. We demonstrated that mTOR and TrxR inhibitors cooperated to induce cell death by triggering oxidative stress, which led to activation of autophagy, endoplasmic reticulum (ER) stress and c-Jun N-terminal Kinase (JNK) signaling pathway in cancer cells. Remarkably, we found that auranofin (AF) combined with everolimus significantly suppressed tumor growth in HCT116 and SGC-7901 xenograft models with no significant signs of toxicity. Conclusion: Our findings identify a promising therapeutic combination for cancer and has important implications for developing mTOR inhibitor-based combination treatments.
    Keywords:  autophagy; c-Jun N-terminal Kinase; mTOR; oxidative stress; thioredoxin reductase
  4. Oncogene. 2021 Mar 25.
      Although epidermal growth factor receptor (EGFR)-targeted therapies are approved for colorectal cancer (CRC) treatment, only 15% of CRC patients respond to EGFR inhibition. Here, we show that colorectal cancers (CRC) can initiate and grow faster through an EGFR-independent mechanism, irrespective of the presence of EGFR, in two different mouse models using tissue-specific ablation of Egfr. The growth benefit in the absence of EGFR is also independent of Kras status. An EGFR-independent gene expression signature, also observed in human CRCs, revealed that anergy-inducing genes are overexpressed in EGFR-independent polyps, suggesting increased infiltration of anergic lymphocytes promotes an accelerated growth rate that is partially caused by escape from cell-mediated immune responses. Many genes in the EGFR-independent gene expression signature are downstream targets of interleukin 10 receptor alpha (IL10RA). We further show that IL10 is detectable in serum from mice with EGFR-independent colon polyps. Using organoids in vitro and Src ablation in vivo, we show that IL10 contributes to growth of EGFR-independent CRCs, potentially mediated by the well-documented role of SRC in IL10 signaling. Based on these data, we show that the combination of an EGFR inhibitor with an anti-IL10 neutralizing antibody results in decreased cell proliferation in organoids and in decreased polyp size in pre-clinical models harboring EGFR-independent CRCs, providing a new therapeutic intervention for CRCs resistant to EGFR inhibitor therapies.
  5. Mol Cell. 2021 Mar 19. pii: S1097-2765(21)00178-7. [Epub ahead of print]
      Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.
    Keywords:  MXD2; Protein translation; S6K1; WTAP; YTHDF readers; cMyc; eIF4A; m(6)A mRNA modification; mRNA stability; mTORC1
  6. Mol Cancer Res. 2021 Mar 22. pii: molcanres.MCR-20-0867-E.2020. [Epub ahead of print]
      AP-2alpha (encoded by TFAP2A) functions as a tumor suppressor and influences response to therapy in several cancer types. We aimed to characterize regulation of the transcriptome by AP-2alpha in colon cancer. CRISPR-Cas9 and shRNA were used to eliminate TFAP2A expression in HCT116 and a panel of colon cancer cell lines. AP-2alpha target genes were identified with RNA-seq and ChIP- seq. Effects on cell cycle were characterized in cells synchronized with aphidicolin and analyzed by FACS and Premo FUCCI. Effects on invasion and tumorigenesis were determined by invasion assay, growth of xenografts and phosphorylated histone H3 (PHH3). Knockout of TFAP2A induced significant alterations in the transcriptome including repression of TGM2, identified as a primary gene target of AP-2alpha. Loss of AP-2alpha delayed progression through S-phase into G2/M and decreased phosphorylation of AKT, effects that were mediated through regulation of TGM2. Buparlisib (BKM120) repressed in vitro invasiveness of HCT116 and a panel of colon cancer cell lines; however, loss of AP-2alpha induced resistance to Buparlisib. Similarly, Buparlisib repressed PHH3 and growth of tumor xenografts and increased overall survival of tumor-bearing mice, whereas, loss of AP-2alpha induced resistance to the effect of PI3K inhibition. Loss of AP-2alpha in colon cancer leads to prolonged S-phase through altered activation of AKT leading to resistance to the PI3K inhibitor, Buparlisib. The findings demonstrate an important role for AP-2alpha in regulating progression through the cell cycle and indicates that AP-2alpha is a marker for response to PI3K inhibitors. Implications: AP-2alpha regulated cell cycle through the PI3K cascade and activation of AKT mediated through TGM2. AP-2alpha induced sensitivity to Buparlisib/BKM120, indicating that AP-2alpha is a biomarker predictive of response to PI3K inhibitors.
  7. Cancer Lett. 2021 Mar 18. pii: S0304-3835(21)00119-1. [Epub ahead of print]507 89-96
      The paucity of targeted treatments available in patients with RAS mutant colorectal cancers contributes to the poor prognosis of this patient group compared to those with RAS wild-type disease. Recent liquid biopsy-driven studies have demonstrated that RAS mutant clones might disappear in plasma during the clonal evolution of the disease, opening new unforeseen perspectives for EGFR blockade in these patients. Nevertheless, the lack of detection of RAS mutations in plasma might depend on the low amount of released circulating tumor DNA (ctDNA), making it necessary a more accurate selection of patients with true RAS mutation conversions. In this liquid biopsy-based study, we assessed RAS mutational status in initially RAS-mutant patients at the time of progressive disease from any line of therapy and investigated the incidence of true conversions to plasma RAS wild-type, comparing a colon cancer specific methylation profile with a mutational signature of ctDNA. Globally, considering either mutational panel or methylation profile as reliable tests to confirm or exclude the presence of ctDNA, the percentage of "true RAS converters" was 37.5%. In our series we observed a trend toward a better PFS in patients who received anti-EGFR as second or subsequent treatment lines compared to those who did not.
    Keywords:  Circulating tumor DNA; Colorectal cancer; EGFR blockade; Methylation; RAS conversion
  8. Clin Colorectal Cancer. 2021 Feb 19. pii: S1533-0028(21)00017-7. [Epub ahead of print]
      BACKGROUND: Colorectal cancer is one of the most common malignancies in both men and women. Despite progress in the treatment of the disease, metastatic colorectal cancer remains lethal with a median survival slightly surpassing 2 years and commonly for some cases a more aggressive course. New therapies are urgently needed based on a better understanding of the molecular pathogenesis of the disease.METHODS: The focus of this investigation is the PIK3CA gene, encoding the alpha catalytic subunit of the enzyme phosphatidylinositol-3 kinase (PI3K). Publicly available data from 3 extensive published series of colorectal carcinomas were analyzed to define the molecular landscape of colorectal adenocarcinomas with and without mutations of PIK3CA. An analysis for discovery of associations with alterations in other critical genes and pathways involved in colorectal cancer was performed. The total mutation burden (TMB) and copy number alteration burden of colorectal cancers with and without mutations of PIK3CA, as well as prognostic implications of alterations of the gene for survival, were examined.
    RESULTS: Mutations in PIK3CA are observed in 20% to 25% of colorectal cancers. PIK3CA represents one of the most frequently mutated oncogenes in these cancers. Mutations in PIK3CA are associated with higher rates of mutations in other genes of important cancer-associated pathways such as the tyrosine kinase receptors/K-Ras/BRAF/MAPK and the Wnt/β-catenin pathway. In addition, PIK3CA mutated colorectal cancers display a higher TMB than nonmutated cancers.
    CONCLUSION: Frequent mutations of PIK3CA gene in colorectal carcinomas may represent an opportunity for targeted therapy combination development inhibiting both the PI3K kinase itself and associated pathway defects. Increased TMB may additionally confer immunotherapy sensitivity, which could be augmented by other targeted therapies.
    Keywords:  Bowel; Catalytic subunit; Molecular lesions; PI3K kinase; Targeted therapies
  9. Mol Cell. 2021 Mar 17. pii: S1097-2765(21)00177-5. [Epub ahead of print]
      The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient, growth, and oncogenic signals. We found that mTORC1 stimulates the synthesis of the major methyl donor, S-adenosylmethionine (SAM), through the control of methionine adenosyltransferase 2 alpha (MAT2A) expression. The transcription factor c-MYC, downstream of mTORC1, directly binds to intron 1 of MAT2A and promotes its expression. Furthermore, mTORC1 increases the protein abundance of Wilms' tumor 1-associating protein (WTAP), the positive regulatory subunit of the human N6-methyladenosine (m6A) RNA methyltransferase complex. Through the control of MAT2A and WTAP levels, mTORC1 signaling stimulates m6A RNA modification to promote protein synthesis and cell growth. A decline in intracellular SAM levels upon MAT2A inhibition decreases m6A RNA modification, protein synthesis rate, and tumor growth. Thus, mTORC1 adjusts m6A RNA modification through the control of SAM and WTAP levels to prime the translation machinery for anabolic cell growth.
    Keywords:  Cell growth; MAT2A; Methionine cycle; N(6)-methyladenosine; Protein Synthesis; RNA metabolism; S-adenosylmethionine; WTAP; mTOR; mTORC1
  10. ESMO Open. 2021 Mar 18. pii: S2059-7029(21)00050-8. [Epub ahead of print]6(2): 100093
      BACKGROUND: Although the efficacy of trifluridine/tipiracil (FTD/TPI) plus bevacizumab (BEV) against metastatic colorectal cancer (mCRC) has been demonstrated, little is known about its effectiveness upon disease stratification by RAS mutations. In this phase II study, we investigated the efficacy and safety profiles of FTD/TPI in mCRC according to RAS mutation status.PATIENTS AND METHODS: Eligible patients were mCRC refractory or intolerant to all standard therapies other than FTD/TPI and regorafenib. Patients received 4-week cycles of treatment with FTD/TPI (35 mg/m2, twice daily, days 1-5 and 8-12) and bevacizumab (5 mg/kg, days 1 and 15). The primary endpoint was disease control rate (DCR). The null hypothesis of DCR in both RAS wild-type (WT) and mutant (MUT) cohorts was 44%, assuming a one-sided significance level of 5.0%. The necessary sample size was estimated to be 49 patients (target sample size: 50 patients) for each cohort.
    RESULTS: Between January and September 2018, 102 patients were enrolled, and 97 patients fulfilled the eligibility criteria (48 in the RAS WT cohort and 49 in the RAS MUT cohort). DCRs in the RAS WT and MUT cohort were 66.7% [90% confidence interval (CI), 53.9%-77.8%, P = 0.0013] and 55.1% (90% CI, 42.4%-67.3%, P = 0.0780), respectively. The median progression-free survival (PFS) and overall survival (OS) were 3.8 and 9.3 months, respectively, in the RAS WT cohort and 3.5 and 8.4 months, respectively, in the RAS MUT cohort. The most common grade 3 or higher adverse event in both cohorts was neutropenia (46% in the RAS WT cohort and 62% in the RAS MUT cohort), without unexpected safety signals.
    CONCLUSIONS: FTD/TPI plus bevacizumab showed promising activity with an acceptable safety profile for pretreated mCRC, regardless of RAS mutation status, although the efficacy outcomes tended to be better in RAS WT.
    Keywords:  JFMC51-1702-C7 trial; RAS mutation status; bevacizumab; metastatic colorectal cancer; trifluridine/tipiracil
  11. Nat Commun. 2021 03 22. 12(1): 1808
      Mutational activation of KRAS promotes the initiation and progression of cancers, especially in the colorectum, pancreas, lung, and blood plasma, with varying prevalence of specific activating missense mutations. Although epidemiological studies connect specific alleles to clinical outcomes, the mechanisms underlying the distinct clinical characteristics of mutant KRAS alleles are unclear. Here, we analyze 13,492 samples from these four tumor types to examine allele- and tissue-specific genetic properties associated with oncogenic KRAS mutations. The prevalence of known mutagenic mechanisms partially explains the observed spectrum of KRAS activating mutations. However, there are substantial differences between the observed and predicted frequencies for many alleles, suggesting that biological selection underlies the tissue-specific frequencies of mutant alleles. Consistent with experimental studies that have identified distinct signaling properties associated with each mutant form of KRAS, our genetic analysis reveals that each KRAS allele is associated with a distinct tissue-specific comutation network. Moreover, we identify tissue-specific genetic dependencies associated with specific mutant KRAS alleles. Overall, this analysis demonstrates that the genetic interactions of oncogenic KRAS mutations are allele- and tissue-specific, underscoring the complexity that drives their clinical consequences.
  12. Theranostics. 2021 ;11(9): 4011-4029
      Rationale: Adenylosuccinate lyase (ADSL) is an essential enzyme for de novo purine biosynthesis. Here we sought to investigate the putative role of ADSL in colorectal carcinoma (CRC) carcinogenesis and response to antimetabolites. Methods: ADSL expression levels were assessed by immunohistochemistry or retrieved from The Cancer Genome Atlas (TCGA) dataset. The effects of ADSL silencing or overexpression were evaluated on CRC cell proliferation, cell migration and cell-cycle. In vivo tumor growth was assessed by the chicken chorioallantoic membrane (CAM). Transfected cell lines or patient-derived organoids (PDO) were treated with 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) and drug response was correlated with ADSL expression levels. Metabolomic and transcriptomic profiling were performed to identify dysregulated pathways and ADSL downstream effectors. Mitochondrial respiration and glycolytic capacity were measured using Seahorse; mitochondrial membrane potential and the accumulation of ROS were measured by FACS using MitoTracker Red and MitoSOX staining, respectively. Activation of canonical pathways was assessed by immunohistochemistry and immunoblotting. Results: ADSL expression is significantly increased in CRC tumors compared to non-tumor tissue. ADSL-high CRCs show upregulation of genes involved in DNA synthesis, DNA repair and cell cycle. Accordingly, ADSL overexpression accelerated progression through the cell cycle and significantly increased proliferation and migration in CRC cell lines. Additionally, ADSL expression increased tumor growth in vivo and sensitized CRCs to 6-MP in vitro, ex vivo (PDOs) and in vivo (CAM model). ADSL exerts its oncogenic function by affecting mitochondrial function via alteration of the TCA cycle and impairment of mitochondrial respiration. The KEAP1-NRF2 and mTORC1-cMyc axis are independently activated upon ADSL overexpression and may favor the survival and proliferation of ROS-accumulating cells, favoring DNA damage and tumorigenesis. Conclusions: Our results suggest that ADSL is a novel oncogene in CRC, modulating mitochondrial function, metabolism and oxidative stress, thus promoting cell cycle progression, proliferation and migration. Our results also suggest that ADSL is a predictive biomarker of response to 6-mercaptopurine in the pre-clinical setting.
    Keywords:  ADSL; colorectal cancer; fumarate; mTOR-MYC-axis; mitochondria
  13. Arch Pharm Res. 2021 Mar 22.
      The transcription factor nuclear factor erythroid 2-like 2 (NEF2L2; NRF2) plays crucial roles in the defense system against electrophilic or oxidative stress by upregulating an array of genes encoding antioxidant proteins, electrophile/reactive oxygen species (ROS) detoxifying enzymes, and drug efflux transporters. In contrast to the protective roles in normal cells, the multifaceted role of NRF2 in tumor growth and progression, resistance to therapy and intratumoral stress, and metabolic adaptation is rapidly expanding, and the complex association of NRF2 with cancer signaling networks is being unveiled. In particular, the implication of NRF2 signaling in cancer stem cells (CSCs), a small population of tumor cells responsible for therapy resistance and tumor relapse, is emerging. Here, we described the dark side of NRF2 signaling in cancers discovered so far. A particular focus was put on the role of NRF2 in CSCs maintenance and therapy resistance, showing that low ROS levels and refractory drug response of CSCs are mediated by the activation of NRF2 signaling. A better understanding of the roles of the NRF2 pathway in CSCs will allow us to develop a novel therapeutic approach to control tumor relapse after therapy.
    Keywords:  Cancer; Cancer stem cell; KEAP1; NRF2; Therapy resistance