bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2021‒08‒15
nineteen papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research

  1. Proc Natl Acad Sci U S A. 2021 Aug 17. pii: e2103261118. [Epub ahead of print]118(33):
      RAS proteins are molecular switches that interact with effector proteins when bound to guanosine triphosphate, stimulating downstream signaling in response to multiple stimuli. Although several canonical downstream effectors have been extensively studied and tested as potential targets for RAS-driven cancers, many of these remain poorly characterized. In this study, we undertook a biochemical and structural approach to further study the role of Sin1 as a RAS effector. Sin1 interacted predominantly with KRAS isoform 4A in cells through an atypical RAS-binding domain that we have characterized by X-ray crystallography. Despite the essential role of Sin1 in the assembly and activity of mTORC2, we find that the interaction with RAS is not required for these functions. Cells and mice expressing a mutant of Sin1 that is unable to bind RAS are proficient for activation and assembly of mTORC2. Our results suggest that Sin1 is a bona fide RAS effector that regulates downstream signaling in an mTORC2-independent manner.
    Keywords:  KRAS; RAS; RBD; Sin1; mTORC2
  2. Expert Opin Biol Ther. 2021 Aug 11.
      INTRODUCTION: : The high frequency of RAS mutations, particularly KRAS mutations, in colorectal cancer (CRC) and the ineffectiveness of anti-EGFR antibodies in treating this disease has created a significant unmet medical need, especially for treating patients in the metastatic phase of this disease. There are many different types of RAS mutations, the most frequent being G12V (c.35G>T (p.G12V)), G12D (c.35G>A (p.G12D)), and G13D (c.38G>A (p.G13D)). Here, we provide an overview of RAS mutations in CRC and their therapeutic implications.AREAS COVERED: : The therapeutic strategies against metastatic CRC with RAS mutations are elaborated according to patient and disease characteristics and integrated into a multiline strategy. The complexity of the molecular structure of RAS and its relationship with the MAPK/ERK pathway partly explain the initial therapeutic failure with MEK or farnesyltransferase inhibitors. Conversely, the development of direct KRAS inhibitors or drugs targeting RAS regulators (e.g., SOS1 and SHP2) has opened new therapeutic fields, requiring the distinction of each KRAS mutation type.
    EXPERT OPINION: In the future, KRAS inhibitors, including SOS1 and SHP2 inhibitors, might be used in combination with other signal transduction inhibitors, such as MEK inhibitors or anti-EGFR antibodies, which block alternative pathways of activation.
    Keywords:  KRAS inhibitors; RAS mutations; SHP2; SOS1; colorectal cancer
  3. Cancer Discov. 2020 Jan;10(1): OF6
      Cis double PIK3CA mutations are common in cancers and increase sensitivity to PI3Kα inhibitors.
  4. Am J Transl Res. 2021 ;13(7): 7458-7474
      Mutation-activated Kras in cancer cells is a well-known challenging treatment-resistant factor that plays a critical role in treatment resistance. Human colorectal cancer (CRC) has four major Kras mutations; KrasG12D (34.2%), KrasG12V (21%), KrasG13D (20%) and KrasG12C (8.4%). Here, we report that while FL118 (a novel inhibitor of survivin, Mcl-1, XIAP, cIAP2 and MdmX) exhibits high efficacy to kill CRC cells and eliminate CRC tumors, CRC cells/tumors with different Kras mutation subtypes in the defined p53/APC genetic statuses exhibit different sensitivity to FL118 treatment. Using CRC cell lines, SW620 (KrasG12V, mutant p53, mutant APC), DLD-1 (KrasG13D, wild type p53, mutant APC) and SNU-C2B (KrasG12D, mutant p53, wild type APC), we demonstrated that silencing of KrasG12V and KrasG12D using Kras-specific shRNA significantly increased CRC cell IC50, while silencing of KrasG13D decreased the CRC cell IC50. This finding suggests that both KrasG12V and KrasG12D are required for showing higher FL118 efficacy, while the presence of KrasG13D could somehow decrease FL118 efficacy under the defined p53/APC genetic status. Consistent with this notion, silencing of KrasG12V in SW620 cells decreased FL118-induced apoptosis, while silencing of KrasG13D in DLD-1 cells increased the FL118-induced apoptosis. Furthermore, forced expression of KrasG12V in SW620 cells increased FL118-induced apoptosis, while forced expression of KrasG13D in DLD-1 cells decreased FL118-induced apoptosis. Additionally, FL118 induced differential reactive oxygen species (ROS) production in SW620, DLD-1 and SNU-C2B cells. Our in vivo studies in animal models further confirmed that SW620 tumors are the most sensitive tumor to FL118 treatment; SNU-C2B tumors are the second most sensitive tumor to FL118 treatment; and the DLD-1 tumors are the least sensitive tumor. These findings would be useful for predicting FL118 sensitivity to patients' CRC tumors with the defined Kras mutation subtypes under the defined p53/APC genetic status.
    Keywords:  FL118; Mcl-1; MdmX; Mutant Kras; XIAP; cIAP2; colorectal cancer; human tumor animal model; survivin
  5. Proc Natl Acad Sci U S A. 2021 Aug 17. pii: e2101496118. [Epub ahead of print]118(33):
      The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.
    Keywords:  Akt; PIP3; kinase; lipid; signaling
  6. Cancer Cell. 2021 Aug 09. pii: S1535-6108(21)00388-3. [Epub ahead of print]39(8): 1059-1061
      RAS mutant tumors have been largely refractory to therapies until now. Recent findings published in the New England Journal of Medicine show that sotorasib provides clinical benefit for KRAS p.G12C-mutated non-small-cell lung cancer (NSCLC) and provide mechanistic insights into acquired resistance to KRASG12C-specific inhibition.
  7. Curr Opin Struct Biol. 2021 Aug 05. pii: S0959-440X(21)00100-7. [Epub ahead of print]71 180-192
      Mutations of RAS genes drive cancer more frequently than any other oncogene. RAS proteins integrate signals from a wide array of receptors and initiate downstream signaling through pathways that control cellular growth. RAS proteins are fundamentally binary molecular switches in which the off/on state is determined by the binding of GDP or GTP, respectively. As such, the intrinsic and regulated nucleotide-binding and hydrolytic properties of the RAS GTPase were historically believed to account for the entirety of the regulation of RAS signaling. However, it is increasingly clear that RAS proteins are also regulated by a vast array of post-translational modifications (PTMs). The current challenge is to understand what are the functional consequences of these modifications and which are physiologically relevant. Because PTMs are catalyzed by enzymes that may offer targets for drug discovery, the study of RAS PTMs has been a high priority for RAS biologists.
    Keywords:  Cancer; GTPase; Post-translational modification; RAS; Signaling
  8. Cancer Discov. 2020 Feb;10(2): OF10
      KRAS4A, but not KRAS4B, directly interacts with hexokinase 1 (HK1) on outer mitochondrial membranes.
  9. Bioengineered. 2021 Dec;12(1): 5099-5109
      Colorectal cancer (CRC) is one of the most important causes of morbidity and mortality in the developed world and is gradually more frequent in the developing world including Saudi Arabia. According to the Saudi Cancer Registry report 2015, CRC is the most common cancer in men (14.9%) and the second most prevalent cancer. Oncogenic mutations in the KRAS gene play a central role in tumorigenesis and are mutated in 30-40% of all CRC patients. To explore the prevalence of KRAS gene mutations in the Saudi population, we collected 80 CRC tumor tissues and sequenced the KRAS gene using automated sequencing technologies. The chromatograms presented mutations in 26 patients (32.5%) in four different codons, that is, 12, 13, 17, and 31. Most of the mutations were identified in codon 12 in 16 patients (61.5% of all mutations). We identified a novel mutation c.51 G>A in codon 17, where serine was substituted by arginine (S17R) in four patients. We also identified a very rare mutation, c.91 G>A, in which glutamic acid was replaced by lysine (E31K) in three patients. In conclusion, our findings further the knowledge about KRAS mutations in different ethnic groups is indispensable to fully understand their role in the development and progression of CRC.
    Keywords:  Colorectal cancer; DNA sequencing; KRAS; Novel mutation; Saudi population
  10. Mol Cancer Ther. 2021 Aug 10. pii: molcanther.1017.2020. [Epub ahead of print]
      Developing effective treatments for colorectal cancers through combinations of small-molecule approaches and immunotherapies present intriguing possibilities for managing these otherwise intractable cancers. During a broad-based, screening effort against multiple colorectal cancer cell lines, we identified indole-substituted quinolines (ISQs), such as N7,N7-dimethyl-3-(1-methyl-1H-indol-3-yl)quinoline-2,7-diamine (ISQ-1), as potent in vitro inhibitors of several cancer cell lines. We found that ISQ-1 inhibited Wnt signaling, a main driver in the pathway governing colorectal cancer development, and ISQ-1 also activated adenosine monophosphate kinase (AMPK), a cellular energy-homeostasis master regulator. We explored the effect of ISQs on cell metabolism. Seahorse assays measuring oxygen consumption rate (OCR) indicated that ISQ-1 inhibited complex I (i.e., NADH ubiquinone oxidoreductase) in the mitochondrial, electron transport chain (ETC). In addition, ISQ-1 treatment showed remarkable synergistic depletion of oncogenic c-Myc protein level in vitro and induced strong tumor remission in vivo when administered together with BI2536, a polo-like kinase-1 (Plk1) inhibitor. These studies point toward the potential value of dual drug therapies targeting the ETC and Plk-1 for the treatment of c-Myc-driven cancers.
  11. Biophys J. 2021 Aug 09. pii: S0006-3495(21)00654-8. [Epub ahead of print]
      KRAS4B is a membrane-anchored signaling protein and a primary target in cancer research. Predictions from molecular dynamics simulations that have previously shaped our mechanistic understanding of KRAS signaling disagree with recent experimental results from neutron reflectometry, nuclear magnetic resonance, and thermodynamic binding studies. To gain insight into these discrepancies, we compare this body of biophysical data to back-calculated experimental results from a series of molecular simulations that implement different subsets of molecular interactions. Our results show that KRAS4B approximates an entropic ensemble of configurations at model membranes containing 30% phosphatidylserine lipids, which is not significantly shaped by interactions between the globular G-domain of KRAS4B and the lipid membrane. These findings revise our understanding of KRAS signaling and promote a model in which the protein samples the accessible conformational space in a near-uniform manner while being available to bind to effector proteins.
  12. EMBO Mol Med. 2021 Aug 08. e13193
      KRAS oncoprotein is commonly mutated in human cancer, but effective therapies specifically targeting KRAS-driven tumors remain elusive. Here, we show that combined treatment with fibroblast growth factor receptor 1 (FGFR1) and polo-like kinase 1 (PLK1) inhibitors evoke synergistic cytotoxicity in KRAS-mutant tumor models in vitro and in vivo. Pharmacological and genetic suppression of FGFR1 and PLK1 synergizes to enhance anti-proliferative effects and cell death in KRAS-mutant lung and pancreatic but not colon nor KRAS wild-type cancer cells. Mechanistically, co-targeting FGFR1 and PLK1 upregulates reactive oxygen species (ROS), leading to oxidative stress-activated c-Jun N-terminal kinase (JNK)/p38 pathway and E2F1-induced apoptosis. We further delineate that autophagy protects from PLK1/FGFR1 inhibitor cytotoxicity and that antagonizing the compensation mechanism by clinically approved chloroquine fully realizes the therapeutic potential of PLK1 and FGFR1 targeting therapy, producing potent and durable responses in KRAS-mutant patient-derived xenografts and a genetically engineered mouse model of Kras-induced lung adenocarcinoma. These results suggest a previously unappreciated role for FGFR1 and PLK1 in the surveillance of metabolic stress and demonstrate a synergistic drug combination for treating KRAS-mutant cancer.
    Keywords:  KRAS-mutant cancer; autophagy; fibroblast growth factor receptor 1; polo-like kinase 1; synthetic lethal vulnerability
  13. Nat Commun. 2021 08 10. 12(1): 4814
      Glutamoptosis is the induction of apoptotic cell death as a consequence of the aberrant activation of glutaminolysis and mTORC1 signaling during nutritional imbalance in proliferating cells. The role of the bioenergetic sensor AMPK during glutamoptosis is not defined yet. Here, we show that AMPK reactivation blocks both the glutamine-dependent activation of mTORC1 and glutamoptosis in vitro and in vivo. We also show that glutamine is used for asparagine synthesis and the GABA shunt to produce ATP and to inhibit AMPK, independently of glutaminolysis. Overall, our results indicate that glutamine metabolism is connected with mTORC1 activation through two parallel pathways: an acute alpha-ketoglutarate-dependent pathway; and a secondary ATP/AMPK-dependent pathway. This dual metabolic connection between glutamine and mTORC1 must be considered for the future design of therapeutic strategies to prevent cell growth in diseases such as cancer.
  14. Cancer Discov. 2020 Jul;10(7): 902
      Colorectal cancer cells lost biosynthetic capabilities in an irreversible differentiation process.
  15. Oncogene. 2021 Aug 12.
      N-myc downstream-regulated gene 1 (NDRG1) is a key regulator that interacts with many classic tumor signaling pathways, including some molecules downstream of the epidermal growth factor receptor (EGFR). However, whether NDRG1 is involved in the mechanism of resistance to cetuximab (CTX), the first monoclonal antibody targeting the EGFR has not been reported. Here, we found that NDRG1 enhanced the sensitivity of CTX in colorectal cancer (CRC) cell lines. Afterwards, we determined the underlying mechanism of this phenomenon. We demonstrated that NDRG1 inhibited the expression of EGFR; blocked EGFR phosphorylation and reduced the EGFR distribution in the cell membrane, cytoplasm and nucleus. And then, NDRG1 suppressed the EGFR downstream signaling: RAS/RAF/ERK and PI3k/AKT/mTOR pathways. Moreover, we discovered that NDRG1 attenuated the endocytosis and degradation of EGFR induced by caveolin-1 (Cav1). Additionally, our findings were further observed in an animal model and human tissues. Our results represent a potentially significant discovery that explains the mechanisms of NDRG1 in CTX resistance. NDRG1 could be a promising biomarker to predict optimum responses to CTX, and a key target to enhance CTX activity in the treatment of metastatic CRC (mCRC).
  16. Nat Commun. 2021 08 10. 12(1): 4838
      Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.
  17. Oncoimmunology. 2021 ;10(1): 1956173
      Immunotherapy targeting the CD274 (PD-L1)/PDCD1 (PD-1) immune checkpoint axis has emerged as a promising treatment strategy for various cancers. Experimental evidence suggests that phosphatidylinositol-4,5-bisphosphonate 3-kinase (PI3K) signaling may upregulate CD274 expression. Thus, we hypothesized that PIK3CA mutation, PTEN loss, or their combined status might be associated with CD274 overexpression in colorectal carcinoma. We assessed tumor CD274 and PTEN expression by immunohistochemistry and assessed PIK3CA mutation by pyrosequencing in 753 patients among 4,465 incident rectal and colon cancer cases that had occurred in two U.S.-wide prospective cohort studies. To adjust for potential confounders and selection bias due to tissue availability, inverse probability weighted multivariable ordinal logistic regression analyses used the 4,465 cases and tumoral data including microsatellite instability, CpG island methylator phenotype, KRAS and BRAF mutations. PIK3CA mutation and loss of PTEN expression were detected in 111 of 753 cases (15%) and 342 of 585 cases (58%), respectively. Tumor CD274 expression was negative in 306 (41%), low in 195 (26%), and high in 252 (33%) of 753 cases. PTEN loss was associated with CD274 overexpression [multivariable odds ratio (OR) 1.83; 95% confidence interval (CI), 1.22-2.75; P = .004]. PIK3CA mutation was statistically-insignificantly (P = .036 with the stringent alpha level of 0.005) associated with CD274 overexpression (multivariable OR, 1.54; 95% CI, 1.03-2.31). PIK3CA-mutated PTEN-lost tumors (n = 33) showed higher prevalence of CD274-positivity (82%) than PIK3CA-wild-type PTEN-lost tumors (n = 204; 70% CD274-positivity) and PTEN-expressed tumors (n = 147; 50% CD274-positivity) (P = .003). Our findings support the role of PI3K signaling in the CD274/PDCD1 pathway.
    Keywords:  Colorectal neoplasms; PI3K pathway; immune checkpoint; molecular pathological epidemiology; tumor microenvironment
  18. Gastric Cancer. 2021 Aug 09.
      The tumor suppressor gene phosphatase and tensin homolog (PTEN) in PI3K/Akt/mTOR pathway is essential in inhibiting tumor growth and metastasis. However, whether the mutation of PTEN gene could induce tumorigenesis and impact the treatment of gastric cancer is still unclear. The purpose of the study was to investigate the combined treatment of gastric tumorigenesis using Rapamycin and Fluorouracil (5-Fu) through interfering with the Akt/mTOR pathway in a mouse model with PTEN conditional deletion. Three groups of mice were exposed for 5 days to Rapamycin and 5-Fu separately and together. The gene expression of the Akt/mTOR pathway, the protein expression of caspase-3 and p-Akt, p-S6K and p-4EBP1, and the pathological changes in stomachs were analyzed. Our study demonstrates that the conditional PTEN deletion in the cells of glandular stomach induces hyperplastic gastric tumors in mice. The combined Rapamycin administration with 5-Fu resulted in better outcomes than their separate administration for the treatment of gastric cancer by inhibiting the mTOR signal pathway. Our study indicates that Rapamycin has a synergistic interaction with chemotherapeutic 5-Fu, and demonstrates a potential therapeutic combination treatment on glandular stomach tumor with PTEN functional absence or aberrantly activated Akt/mTOR pathway. It provides important insights into the inhibition of the Akt/mTOR pathway in gastric cancer clinical therapy.
    Keywords:  5-Fluorouracil; Akt/mTOR; Gastric tumorigenesis; PTEN; Rapamycin
  19. Front Cell Dev Biol. 2021 ;9 705060
      BRAF mutated colon cancer presents with poor survival, and the treatment strategies are controversial. The tumor microenvironment, which plays a key role in tumorigenesis as well as responses to treatments, of this subtype is largely unknown. In the present study, we analyzed the differences of immune microenvironments between BRAF mutated and BRAF wild-type colon cancer utilizing datasets from The Cancer Genome Atlas and Gene Expression Omnibus and confirmed the findings by tissue specimens of patients. We found that BRAF mutated colon cancer had more stromal cells, more immune cell infiltration, and lower tumor purity. Many immunotherapeutic targets, including PD-1, PD-L1, CTLA-4, LAG-3, and TIM-3, were highly expressed in BRAF mutated patients. BRAF mutation was also correlated with higher proportions of neutrophils and macrophages M1, and lower proportions of plasma cells, dendritic cells resting, and T cells CD4 naïve. In conclusion, our study demonstrates a different pattern of the immune microenvironment in BRAF mutated colon cancer and provides insights into the future use of checkpoint inhibitors in this subgroup of patients.
    Keywords:  BRAF; colon cancer; immune microenvironment; immunotherapy; therapeutic target