bims-pimaco Biomed News
on PI3K and MAPK signalling in colorectal cancer
Issue of 2021‒04‒04
thirty papers selected by
Lucas B. Zeiger
Beatson Institute for Cancer Research

  1. Nat Cell Biol. 2021 Apr 01.
      Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.
  2. Cells. 2021 Mar 17. pii: 667. [Epub ahead of print]10(3):
      The most commonly mutated isoform of RAS among all cancer subtypes is KRAS. In this review, we focus on the special role of KRAS mutations in colorectal cancer (CRC), aiming to collect recent data on KRAS-driven enhanced cell signalling, in vitro and in vivo research models, and CRC development-related processes such as metastasis and cancer stem cell formation. We attempt to cover the diverse nature of the effects of KRAS mutations on age-related CRC development. As the incidence of CRC is rising in young adults, we have reviewed the driving forces of ageing-dependent CRC.
    Keywords:  CRC with age; KRAS; RAS signalling; RAS-driven metastasis; cancer stem cells; colorectal cancer
  3. FEBS J. 2021 Apr 03.
      Many metabolic phenotypes in cancer cells are also characteristic of proliferating non-transformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate, and provide a model to distinguish the metabolic influences of oncogenic mutations and non-oncogenic growth.
    Keywords:  Cancer metabolism; KRAS; branched-chain amino acids; cell growth; cell proliferation
  4. Growth Factors. 2021 Mar 27. 1-10
      For patients with metastatic colorectal cancer (mCRC), epidermal growth factor receptor (EGFR) inhibitors are limited to patients with RAS wild-type tumours. Not all patients will benefit from treatment and better predictive biomarkers are needed. Here we investigated the prognostic and predictive impact of the EGFR ligands amphiregulin (AREG) and epiregulin (EREG). Expression levels were assessed by immunohistochemistry on 99 KRAS wild-type tumours. AREG and EREG positivity was seen in 49% and 50% of cases, respectively. No difference in expression was observed by primary tumour side. There was no significant difference in OS by AREG or EREG expression. In the subset of patients who received an EGFR inhibitor, EREG positivity was associated with longer OS (median 34.0 vs. 27.0 months, p = 0.033), driven by a difference in patients with a left-sided primary (HR 0.37, p = 0.015). Our study supports further investigation into EREG as a predictive biomarker in mCRC.
    Keywords:  EGFR inhibitor therapy; EGFR ligand; biomarker; cetuximab; metastatic colorectal cancer; panitumumab
  5. Front Cell Dev Biol. 2021 ;9 655889
      Oncogene c-Myc (referred in this report as MYC) promotes tumorigenesis in multiple human cancers. MYC regulates numerous cellular programs involved in cell growth and cell metabolism. Tumor cells exhibit obligatory dependence on cholesterol metabolism, which provides essential membrane components and metabolites to support cell growth. To date, how cholesterol biosynthesis is delicately regulated to promote tumorigenesis remains unclear. Here, we show that MYC enhances cholesterol biosynthesis and promotes cell proliferation. Through transcriptional upregulation of SQLE, a rate-limiting enzyme in cholesterol synthesis pathway, MYC increases cholesterol production and promotes tumor cell growth. SQLE overexpression restores the cellular cholesterol levels in MYC-knockdown cells. More importantly, in SQLE-depleted cells, enforced expression of MYC has no effect on cholesterol levels. Therefore, our findings reveal that SQLE is critical for MYC-mediated cholesterol synthesis, and further demonstrate that SQLE may be a potential therapeutic target in MYC-amplified cancers.
    Keywords:  MYC; SQLE; cancer; cell proliferation; cholesterol synthesis
  6. Biomolecules. 2021 Mar 02. pii: 370. [Epub ahead of print]11(3):
      BACKGROUND: The oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation was reported to be the signature genetic event in most cases of pancreatic ductal adenocarcinoma (PDAC). Hepassocin (HPS/FGL1) is involved in regulating lipid metabolism and the progression of several cancer types; however, the underlying mechanism of HPS/FGL1 in the KRAS mutant PDAC cells undergoing eicosapentaenoic acid (EPA) treatment remains unclear.METHODS: We measured HPS/FGL1 protein expressions in a human pancreatic ductal epithelial (HPNE) normal pancreas cell line, a KRAS-wild-type PDAC cell line (BxPC-3), and KRAS-mutant PDAC cell lines (PANC-1, MIA PaCa-2, and SUIT-2) by Western blot methods. HEK293T cells were transiently transfected with corresponding KRAS-expressing plasmids to examine the level of HPS expression with KRAS activation. We knocked-down HPS/FGL1 using lentiviral vectors in SUIT-2 cells and measured the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenicity assays. Furthermore, a lipidomic analysis was performed to profile changes in lipid metabolism after HPS/FGL1 knockdown.
    RESULTS: We found that the HPS/FGL1 level was significantly upregulated in KRAS-mutated PDAC cells and was involved in KRAS/phosphorylated (p)-signal transduction and activator of transcription 3 (STAT3) signaling, and the knockdown of HPS/FGL1 in SUIT-2 cells decreased cell proliferation through increasing G2/M cell cycle arrest and cyclin B1 expression. In addition, the knockdown of HPS/FGL1 in SUIT-2 cells significantly increased omega-3 polyunsaturated fatty acids (PUFAs) and EPA production but not docosahexaenoic acid (DHA). Moreover, EPA treatment in SUIT-2 cells reduced the expression of de novo lipogenic protein, acetyl coenzyme A carboxylase (ACC)-1, and decreased p-STAT3 and HPS/FGL1 expressions, resulting in the suppression of cell viability.
    CONCLUSIONS: Results of this study indicate that HPS is highly expressed by KRAS-mutated PDAC cells, and HPS/FGL1 plays a crucial role in altering lipid metabolism and increasing cell growth in pancreatic cancer. EPA supplements could potentially inhibit or reduce ACC-1-involved lipogenesis and HPS/FGL1-mediated cell survival in KRAS-mutated pancreatic cancer cells.
    Keywords:  KRAS mutation; eicosapentaenoic acid; hepassocin; pancreatic cancer
  7. Gut. 2021 Mar 30. pii: gutjnl-2020-322660. [Epub ahead of print]
      BACKGROUND: Gastric adenocarcinoma (GAC) is a lethal disease with limited therapeutic options. Genetic alterations in chromatin remodelling gene AT-rich interactive domain 1A (ARID1A) and mTOR pathway activation occur frequently in GAC. Targeting the mechanistic target of rapamycin (mTOR) pathway in unselected patients has failed to show survival benefit. A deeper understanding of GAC might identify a subset that can benefit from mTOR inhibition.METHODS: Genomic alterations in ARID1A were analysed in GAC. Mouse gastric epithelial cells from CK19-Cre-Arid1Afl/fl and wild-type mice were used to determine the activation of oncogenic genes due to loss of Arid1A. Functional studies were performed to determine the significance of loss of ARID1A and the sensitivity of ARID1A-deficient cancer cells to mTOR inhibition in GAC.
    RESULTS: More than 30% of GAC cases had alterations (mutations or deletions) of ARID1A and ARID1A expression was negatively associated with phosphorylation of S6 and SOX9 in GAC tissues and patient-derived xenografts (PDXs). Activation of mTOR signalling (increased pS6) and SOX9 nuclear expression were strongly increased in Arid1A-/- mouse gastric tissues which could be curtailed by RAD001, an mTOR inhibitor. Knockdown of ARID1A in GAC cell lines increased pS6 and nuclear SOX9 and increased sensitivity to an mTOR inhibitor which was further amplified by its combination with fluorouracil both in vitro and in vivo in PDXs.
    CONCLUSIONS: The loss of ARID1A activates pS6 and SOX9 in GAC, which can be effectively targeted by an mTOR inhibitor. Therefore, our studies suggest a new therapeutic strategy of clinically targeting the mTOR pathway in patients with GAC with ARID1A deficiency.
    Keywords:  cancer; gastric cancer; gastric neoplasia; gastrointestinal cancer; gene expression
  8. Biomolecules. 2021 Mar 30. pii: 518. [Epub ahead of print]11(4):
      Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK's catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting.
    Keywords:  ERK; KinCon reporter; MAPK pathway; RAF; RAS; RAS–RAF–ERK pathway; allosteric inhibitor; biosensor; cancer mutations; kinase inhibitor; personalized therapy; protein-fragment complementation assay; structure dynamics; structure simulations
  9. Cell Rep. 2021 Mar 30. pii: S2211-1247(21)00242-4. [Epub ahead of print]34(13): 108928
      Flux through the RAF-MEK-ERK protein kinase cascade is shaped by phosphatases acting on the core components of the pathway. Despite being an established drug target and a hub for crosstalk regulation, little is known about dephosphorylation of MEK, the central kinase within the cascade. Here, we identify PPP6C, a phosphatase frequently mutated or downregulated in melanoma, as a major MEK phosphatase in cells exhibiting oncogenic ERK pathway activation. Recruitment of MEK to PPP6C occurs through an interaction with its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting signaling through the ERK pathway and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation, suggesting that they promote disease at least in part by activating the core oncogenic pathway driving melanoma. Collectively, our studies identify a key negative regulator of ERK signaling that may influence susceptibility to targeted cancer therapies.
    Keywords:  MAP kinase signaling; cancer cell signaling; kinase inhibitor resistance; melanoma; protein phosphatases
  10. Front Oncol. 2021 ;11 637298
      Background: The incidence of colorectal cancer (CRC) has been increasing worldwide in recent years. Targeting cancer stem cells (CSCs) in CRC remains a difficult challenge. KDM2B and EZH2 play important role in the maintenance of CSCs' self-renewal capacity and tumorigenic ability; however, the biological functions of those genes in CRC remain unclear. In this study, we aimed to define the contribution of the expression of KDM2B in the features of CRC and establish the relationship between KDM2B and EZH2 in colorectal CSCs. Methods: The expression of KDM2B and EZH2 in the specimens of CRC and CRC cell lines were analyzed by immunohistochemistry, Western blot, and immunofluorescence. The underlying mechanisms of altered expressions of KDM2B and EZH2 and their impact on the biologic features of CRC and stemness in CRC were investigated. Results: The KDM2B gene was highly expressed in CRC tissues, and its overexpression positively correlated with tumor stages and tumor/node/metastasis (TNM) classification. The downregulation of KDM2B retarded cell proliferation, induced DNA damage, reduced spheroid formation, and decreased CRC stem cell markers: CD44, CD133, and ALDH-1. Moreover, the downregulation of KDM2B decreased the expression of EZH2 and both regulated cell migration, invasion, and stemness in the CRC cell line. Additionally, the interaction between KDM2B and EZH2 significantly increased the components of the PI3K/AKT pathway including AKT and PI3K. The high expression of KDM2B positively correlated with EZH2 in CRC tissues. Conclusion: This study shows that the downregulation of KDM2B and EZH2 can regulate CRC cell stemness, and their interaction may serve as a novel prognostic marker and therapeutic target for patients with CRC.
    Keywords:  EZH2; KDM2B; PI3K/AKT; colorectal cancer; stemness
  11. Cancers (Basel). 2021 Mar 12. pii: 1267. [Epub ahead of print]13(6):
      The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.
    Keywords:  cancer drivers; colorectal cancer; copy number alterations; exome sequencing; genomic instability; mouse models; mutational signature; organoids; single cell; single nucleotide variants
  12. Cancers (Basel). 2021 Mar 26. pii: 1538. [Epub ahead of print]13(7):
      The phosphatidylinositol-3-kinase (PI3K) pathway plays a central role in the regulation of several signalling cascades which regulate biological processes such as cellular growth, survival, proliferation, motility and angiogenesis. The hyperactivation of this pathway is linked to tumour progression and is one of the most common events in human cancers. Additionally, aberrant activation of the PI3K pathway has been demonstrated to limit the effectiveness of a number of anti-tumour agents paving the way for the development and implementation of PI3K inhibitors in the clinic. However, the overall effectiveness of these compounds has been greatly limited by inadequate target engagement due to reactivation of the pathway by compensatory mechanisms. Herein, we review the common adaptive responses that lead to reactivation of the PI3K pathway, therapy resistance and potential strategies to overcome these mechanisms of resistance. Furthermore, we highlight the potential role in changes in cellular plasticity and PI3K inhibitor resistance.
    Keywords:  PI3K pathway; PI3K pathway inhibitors; mechanisms of resistance
  13. Zhongguo Zhong Yao Za Zhi. 2021 Mar;46(5): 1197-1204
      To screen the sensitive cell lines of active fraction from clove(AFC) on human colon cancer cells, investigate the effects of AFC on the cells proliferation and apoptosis as well as PI3 K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin) signaling pathways involved, and reveal the mechanism of AFC for inducing apoptosis of human colorectal carcinoma cells. Cell counting kit-8(CCK-8) assay was used to detect the cytotoxic effect of different concentrations of AFC. AFC-induced apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining. HCT116 cells were treated with AFC with or without pretreatment with insulin-like growth factor-Ⅰ(IGF-Ⅰ), and then the protein expression levels of caspase-3, caspase-9, poly ADP-ribose polymerase(PARP), PI3 K, p-PI3 K, Akt, p-Akt, mTOR and p-mTOR in PI3 K/Akt/mTOR signaling pathway were detected by Western blot. RESULTS:: showed that the most obvious inhibitory effect of AFC was on human colon cancer HCT116 cells, and the optimal AFC treatment time was 48 hours. After AFC treatment, typical apoptotic features such as nuclear chromatin concentration, nuclear fragmentation and apoptotic bodies appeared in a dose-dependent manner. Annexin V-FITC/PI double staining showed that as compared with the control group, 50 and 100 μg·mL~(-1) AFC groups increased the apoptosis rate of HCT116 cells significantly(P<0.001); AFC activated caspase-9, cleaved caspase-3 and cleaved PARP in a concentration-dependent manner. The protein expression levels of cleaved caspase-3/procaspase-3, cleaved PARP/PARP and caspase-9/β-actin after treatment of AFC(100 μg·mL~(-1)) were significantly different from those in the control group(P<0.001). The relative protein expression of p-PI3 K, p-Akt and p-mTOR decreased in a concentration dependent manner, while Akt and mTOR showed no significant differences among groups. The ratios of p-PI3 K/PI3 K, p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100 μg·mL~(-1)) were significantly lower than those in the control group(P<0.01). Its combination with IGF-Ⅰ weakened the effect of AFC in inhibiting PI3 K/Akt/mTOR signaling pathway. The ratios of p-Akt/Akt and p-mTOR/mTOR in the AFC+IGF-Ⅰ group were significantly enhanced as compared with the AFC group(P<0.05). Apoptosis-related protein expression levels(cleaved caspase-3 and cleaved PARP) in HCT116 cells treated with AFC+IGF-Ⅰ were also down regulated. As compared with the AFC group, the ratios of cleaved caspase-3/procaspase-3 and cleaved PARP/PARP in the AFC+IGF-Ⅰ group were significantly decreased(P<0.01). In summary, AFC activated caspase-mediated cascades and induced HCT116 cells apoptosis in a dose-dependent manner, which may be associated with the inhibition of the PI3 K/Akt/mTOR signaling pathway.
    Keywords:  PI3K/Akt/mTOR pathway; active fraction from clove; apoptosis; colon cancer
  14. J Exp Clin Cancer Res. 2021 Mar 30. 40(1): 114
      BACKGROUND: Colorectal cancer (CRC) is a clinically challenging malignant tumor worldwide. As a natural product and sesquiterpene lactone, Costunolide (CTD) has been reported to possess anticancer activities. However, the regulation mechanism and precise target of this substance remain undiscovered in CRC. In this study, we found that CTD inhibited CRC cell proliferation in vitro and in vivo by targeting AKT.METHODS: Effects of CTD on colon cancer cell growth in vitro were evaluated in cell proliferation assays, migration and invasion, propidium iodide, and annexin V-staining analyses. Targets of CTD were identified utilizing phosphoprotein-specific antibody array; Costunolide-sepharose conjugated bead pull-down analysis and knockdown techniques. We investigated the underlying mechanisms of CTD by ubiquitination, immunofluorescence staining, and western blot assays. Cell-derived tumour xenografts (CDX) in nude mice and immunohistochemistry were used to assess anti-tumour effects of CTD in vivo.
    RESULTS: CTD suppressed the proliferation, anchorage-independent colony growth and epithelial-mesenchymal transformation (EMT) of CRC cells including HCT-15, HCT-116 and DLD1. Besides, the CTD also triggered cell apoptosis and cell cycle arrest at the G2/M phase. The CTD activates and induces p53 stability by inhibiting MDM2 ubiquitination via the suppression of AKT's phosphorylation in vitro. The CTD suppresses cell growth in a p53-independent fashion manner; p53 activation may contribute to the anticancer activity of CTD via target AKT. Finally, the CTD decreased the volume of CDX tumors without of the body weight loss and reduced the expression of AKT-MDM2-p53 signaling pathway in xenograft tumors.
    CONCLUSIONS: Our project has uncovered the mechanism underlying the biological activity of CTD in colon cancer and confirmed the AKT is a directly target of CTD. All of which These results revealed that CTD might be a new AKT inhibitor in colon cancer treatment, and CTD is worthy of further exploration in preclinical and clinical trials.
    Keywords:  AKT; AKT/MDM2/p53 pathway; Colon cancer; Costunolide (CTD); Ubiquitination, Xenograft model
  15. Phytother Res. 2021 Apr 01.
      Brassinin (BSN), a precursor of phytoalexins, extracted from Chinese cabbage has been reported to act as a promising anti-neoplastic agent. However, the effects of BSN on colon cancer cells and its underlying mechanisms have not been fully elucidated. This study aimed at investigating the anti-neoplastic impact of BSN and its possible synergistic effect with paclitaxel on colon cancer cells. The effect of BSN on Janus-activated kinases (JAKs)/signal transducer and activator of transcription 3 (STAT3) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways and its downstream functions was deciphered using diverse assays in colon carcinoma cells. We found that BSN displayed significant cytotoxic effect and suppressed cell proliferation on colon carcinoma cells. Additionally, it was noted that BSN modulated oncogenic gene expression and induced apoptosis through down regulating multiple oncogenic signaling cascades such as JAKs/STAT3 and PI3K/Akt/mTOR simultaneously. Besides, BSN-paclitaxel combination significantly increased cytotoxicity and induced apoptosis synergistically as compared with individual treatment of both the agents. Overall, our findings indicate that BSN may be a novel candidate for anti-colon cancer targeted therapy.
    Keywords:  JAKs/STAT3; PI3K/Akt/mTOR; brassinin; colorectal cancer cells
  16. EMBO Rep. 2021 Mar 30. e51415
      The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R-spondin induces auto-ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R-spondin by protecting ZNRF3/RNF43 from ubiquitin-dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R-spondin-LGR4-ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R-spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.
    Keywords:  EMT; LGR4/5/6; colorectal cancer; deubiquitination; mouse intestinal organoids
  17. Sci Rep. 2021 Apr 01. 11(1): 7344
      Nectin-4, upregulated in various cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2∆Ex16, enhancing DNA synthesis through the PI3K-AKT signaling in human breast cancer T47D cells in an adherent culture. We found here that nectin-4 and p95-ErbB2, but not nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced SOX2 gene expression and cell proliferation in a suspension culture. This enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2 was dependent on the SOX2 gene expression. In T47D cells, nectin-4 and any one of p95-ErbB2, ErbB2, or ErbB2∆Ex16 cooperatively activated the PI3K-AKT signaling, known to induce the SOX2 gene expression, to similar extents. However, only a combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced the SOX2 gene expression. Detailed studies revealed that only nectin-4 and p95-ErbB2 cooperatively activated the Hippo signaling. YAP inhibited the SOX2 gene expression in this cell line and thus the MST1/2-LATS1/2 signaling-mediated YAP inactivation increased the SOX2 gene expression. These results indicate that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively regulates the Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.
  18. Biophys J. 2021 Mar 25. pii: S0006-3495(21)00248-4. [Epub ahead of print]
      Akt plays a key role in the Ras/PI3K/Akt/mTOR signaling pathway. In breast cancer, Akt translocation to the plasma membrane is enabled by the interaction of its pleckstrin homology domain (PHD) with calmodulin (CaM). At the membrane, the conformational change promoted by PIP3 releases CaM and facilitates Thr308 and Ser473 phosphorylation and activation. Here, using modelling and molecular dynamics (MD) simulations we aim to figure out how CaM interacts with Akt's PHD at the atomic level. Our simulations show that the CaM-PHD interaction is thermodynamically stable and involves a β-strand, rather than an α-helix, in agreement with NMR data, and that electrostatic and hydrophobic interactions are critical. PHD interacts with CaM lobes; however, multiple modes are possible. IP4, the polar head of PIP3, weakens the CaM-PHD interaction, implicating the release mechanism at the plasma membrane. Recently, we unraveled the mechanism of PI3Kα activation at the atomistic level and the structural basis for Ras role in the activation. Here, our atomistic structural data clarify the mechanism of how CaM interacts, delivers, and releases Akt - the next node in the Ras/PI3K pathway - at the plasma membrane.
    Keywords:  Akt activation; CaM; MD Simulation; PH domain; PI3K; Phosphorylation; Ras; plasma membrane; signaling pathway
  19. Front Oncol. 2021 ;11 638360
      Mutations of the proto-oncogene KRAS are the most frequent gain-of-function alterations found in cancer. KRAS is mutated in about 30% of all human tumors, but it could reach more than 90% in certain cancer types such as pancreatic adenocarcinoma. Although historically considered to be undruggable, a particular KRAS mutation, the G12C variant, has recently emerged as an actionable alteration especially in non-small cell lung cancer (NSCLC). KRASG12C and pan-KRAS inhibitors are being tested in clinical trials and have recently shown promising activity. Due to the difficulties in direct targeting of KRAS, other approaches are being explored. The inhibition of target upstream activators or downstream effectors of KRAS pathway has shown to be moderately effective given the evidence of emerging mechanisms of resistance. Various synthetic lethal partners of KRAS have recently being identified and the inhibition of some of those might prove to be successful in the future. The study of escape mechanisms to KRAS inhibition could support the utility of combination strategies in overcoming intrinsic and adaptive resistance and enhancing clinical benefit of KRASG12C inhibitors. Considering the role of the microenvironment in influencing tumor initiation and promotion, the immune tumor niche of KRAS mutant tumors has been deeply explored and characterized for its unique immunosuppressive skewing. However, a number of aspects remains to be fully understood, and modulating this tumor niche might revert the immunoresistance of KRAS mutant tumors. Synergistic associations of KRASG12C and immune checkpoint inhibitors are being tested.
    Keywords:  G12C mutation; KRAS; NSCLC; colon cancer; pancreatic cancer
  20. Biomolecules. 2021 Mar 03. pii: 377. [Epub ahead of print]11(3):
      RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein-protein and protein-lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed.
    Keywords:  RAS; dimers; membrane dynamics; multimers; nanoclusters; nonproductive interfaces
  21. Front Cell Dev Biol. 2021 ;9 647311
      Molecularly tailored therapies have opened a new era, chronic myeloid leukemia being the ideal example, in the treatment of cancer. However, available therapeutic options are still unsatisfactory in many types of cancer, and often fail due to the occurrence of resistance mechanisms. With regard to small-molecule compounds targeting the components of the Mitogen-Activated Protein Kinase (MAPK) cascade RAF-MEK1/2-ERK1/2, these drugs may result ineffective as a consequence of the activation of compensatory pro-survival/proliferative signals, including receptor tyrosine kinases, PI3K, as well as other components of the MAPK family such as TPL2/COT. The MAPK ERK5 has been identified as a key signaling molecule in the biology of several types of cancer. In this review, we report pieces of evidence regarding the activation of the MEK5-ERK5 pathway as a resistance mechanism to RAF-MEK1/2-ERK1/2 inhibitors. We also highlight the known and possible mechanisms underlying the cross-talks between the ERK1/2 and the ERK5 pathways, the characterization of which is of great importance to maximize, in the future, the impact of RAF-MEK1/2-ERK1/2 targeting. Finally, we emphasize the need of developing additional therapeutically relevant MEK5-ERK5 inhibitors to be used for combined treatments, thus preventing the onset of resistance to cancer therapies relying on RAF-MEK1/2-ERK1/2 inhibitors.
    Keywords:  ERK1/2/5; MAPK; cancer; combined therapy; resistance mechanisms; targeted therapy
  22. Sci Rep. 2021 Mar 30. 11(1): 7199
      The disulfide isomerase ERp57, originally found in the endoplasmic reticulum, is located in multiple cellular compartments, participates in diverse cell functions and interacts with a huge network of binding partners. It was recently suggested as an attractive new target for cancer therapy due to its critical role in tumor cell proliferation. Since a major bottleneck in cancer treatment is the occurrence of hypoxic areas in solid tumors, the role of ERp57 in cell growth was tested under oxygen depletion in the colorectal cancer cell line HCT116. We observed a severe growth inhibition when ERp57 was knocked down in hypoxia (1% O2) as a consequence of downregulated c-Myc, PLK1, PDPK1 (PDK1) and AKT (PKB). Further, irradiation experiments revealed also a radiosensitizing effect of ERp57 depletion under oxygen deprivation. Compared to ERp57, we do not favour PDPK1 as a suitable pharmaceutical target as its efficient knockdown/chemical inhibition did not show an inhibitory effect on proliferation.
  23. Cancers (Basel). 2021 Mar 02. pii: 1047. [Epub ahead of print]13(5):
      Epidermal growth factor receptor (EGFR) is one of the anticancer drug targets for certain malignancies, including nonsmall cell lung cancer (NSCLC), colorectal cancer (CRC), and head and neck squamous cell carcinoma. However, the grave issue of drug resistance through diverse mechanisms persists, including secondary EGFR-mutation and its downstream RAS/RAF mutation. Since the discovery of the role of human epidermal growth factor receptor 2 (HER2) and HER3 in drug resistance, HER2- or HER3-targeting treatment strategies using monoclonal antibodies have been intensively examined and have demonstrated impressive responsiveness and limitations. Finally, an innovative targeted therapy called antibody drug conjugates (ADC) has provided a solution to overcome this resistance. Specifically, a new cleavable linker-payload system enables stable drug delivery to cancer cells, causing selective destruction. HER2-targeting ADC trastuzumab deruxtecan demonstrated promising responsiveness in patients with HER2-positive CRC, in a phase 2 clinical trial (objective response rate = 45.3%). Furthermore, HER3-targeting patritumab deruxtecan, another ADC, exhibited impressive tumor shrinkage in pretreated patients with EGFR-mutated NSCLC, in a phase 1 clinical trial. This manuscript presents an overview of the accumulated evidence on HER2- and HER3-targeting therapy, especially ADCs, and discussion of remaining issues for further improving these treatments in cancers resistant to EGFR inhibitors.
    Keywords:  ADC; CRC; EGFR; EGFR-TKI; HER2; HER3; HNSCC; NSCLC; patritumab deruxtecan; trastuzumab deruxtecan (T-DXd)
  24. Nat Commun. 2021 Apr 01. 12(1): 2038
      Wild-type KRAS (KRASWT) amplification has been shown to be a secondary means of KRAS activation in cancer and associated with poor survival. Nevertheless, the precise role of KRASWT overexpression in lung cancer progression is largely unexplored. Here, we identify and characterize a KRAS-responsive lncRNA, KIMAT1 (ENSG00000228709) and show that it correlates with KRAS levels both in cell lines and in lung cancer specimens. Mechanistically, KIMAT1 is a MYC target and drives lung tumorigenesis by promoting the processing of oncogenic microRNAs (miRNAs) through DHX9 and NPM1 stabilization while halting the biogenesis of miRNAs with tumor suppressor function via MYC-dependent silencing of p21, a component of the Microprocessor Complex. KIMAT1 knockdown suppresses not only KRAS expression but also KRAS downstream signaling, thereby arresting lung cancer growth in vitro and in vivo. Taken together, this study uncovers a role for KIMAT1 in maintaining a positive feedback loop that sustains KRAS signaling during lung cancer progression and provides a proof of principle that interfering with KIMAT1 could be a strategy to hamper KRAS-induced tumorigenesis.
  25. Cancers (Basel). 2021 Mar 04. pii: 1097. [Epub ahead of print]13(5):
      Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.
    Keywords:  colorectal cancer; patient-derived cancer models; single-cell RNA-sequencing; transcriptional programs; tumor cell differentiation; tumor heterogeneity; tumor metabolism; tumor-initiating cells
  26. Cancers (Basel). 2021 Mar 10. pii: 1204. [Epub ahead of print]13(6):
      The Kirsten rat sarcoma viral oncogene homolog (KRAS) is mutated in approximately 25% of all human cancers and is known to be a major player promoting and maintaining tumorigenesis through the RAS/MAPK pathway. Over the years, a large number of studies have identified strategies at different regulatory levels to tackle this 'difficult-to-target' oncoprotein. Yet, the most ideal strategy to overcome KRAS and its downstream effects has yet to be uncovered. This review summarizes the role of KRAS activating mutations in multiple cancer types as well as the key findings for potential strategies inhibiting its oncogenic behavior. A comprehensive analysis of the different pathways and mechanisms associated with KRAS activity in tumors will ultimately pave the way for promising future work that will identify optimum therapeutic strategies.
    Keywords:  EGFR; KRAS; MAPK; cancer; mutations; targeted-therapy
  27. Cancers (Basel). 2021 Mar 04. pii: 1092. [Epub ahead of print]13(5):
      The treatment options available for colorectal cancer (CRC) have increased over the years and have significantly improved the overall survival of CRC patients. However, the response rate for CRC patients with metastatic disease remains low and decreases with subsequent lines of therapy. The clinical management of patients with metastatic CRC (mCRC) presents a unique challenge in balancing the benefits and harms while considering disease progression, treatment-related toxicities, drug resistance and the patient's overall quality of life. Despite the initial success of therapy, the development of drug resistance can lead to therapy failure and relapse in cancer patients, which can be attributed to the cancer stem cells (CSCs). Thus, colorectal CSCs (CCSCs) contribute to therapy resistance but also to tumor initiation and metastasis development, making them attractive potential targets for the treatment of CRC. This review presents the available CCSC isolation methods, the clinical relevance of these CCSCs, the mechanisms of drug resistance associated with CCSCs and the ongoing clinical trials targeting these CCSCs. Novel therapeutic strategies are needed to effectively eradicate both tumor growth and metastasis, while taking into account the tumor microenvironment (TME) which plays a key role in tumor cell plasticity.
    Keywords:  cancer stem cells; clinical trials; colorectal cancer; drug resistance
  28. Front Cell Dev Biol. 2021 ;9 626316
      Interpreting connections between the multiple networks of cell metabolism is indispensable for understanding how cells maintain homeostasis or transform into the decontrolled proliferation phenotype of cancer. Situated at a critical metabolic intersection, citrate, derived via glycolysis, serves as either a combustible fuel for aerobic mitochondrial bioenergetics or as a continuously replenished cytosolic carbon source for lipid biosynthesis, an essentially anaerobic process. Therein lies the paradox: under what conditions do cells control the metabolic route by which they process citrate? The Warburg effect exposes essentially the same dilemma-why do cancer cells, despite an abundance of oxygen needed for energy-generating mitochondrial respiration with citrate as fuel, avoid catabolizing mitochondrial citrate and instead rely upon accelerated glycolysis to support their energy requirements? This review details the genesis and consequences of the metabolic paradigm of a "truncated" Krebs/TCA cycle. Abundant data are presented for substrate utilization and membrane cholesterol enrichment in tumors that are consistent with criteria of the Warburg effect. From healthy cellular homeostasis to the uncontrolled proliferation of tumors, metabolic alterations center upon the loss of regulation of the cholesterol biosynthetic pathway. Deregulated tumor cholesterogenesis at the HMGR locus, generating enhanced carbon flux through the cholesterol synthesis pathway, is an absolute prerequisite for DNA synthesis and cell division. Therefore, expedited citrate efflux from cholesterol-enriched tumor mitochondria via the CTP/SLC25A1 citrate transporter is fundamental for sustaining the constant demand for cytosolic citrate that fuels the elevated flow of carbons from acetyl-CoA through the deregulated pathway of cholesterol biosynthesis.
    Keywords:  Warburg effect; mitochondrial citrate export; truncated Krebs/TCA cycle; tumor cholesterogenesis; tumor membrane cholesterol
  29. Front Oncol. 2021 ;11 522899
      Objective: The expression of Wnt7a in colorectal cancer tissues and cell lines was analyzed, and the effect of Wnt7a on the proliferation of colorectal cancer cells was studied, so as to confirm the relationship between Wnt7a and the occurrence and development of colorectal cancer.Methods: (1) Immunohistochemical method was used to compare the expression of Wnt7a in different tissues and its relationship with the clinicopathology of colorectal adenocarcinoma. (2) The expression levels of Wnt7a in colorectal cancer cell lines HT-29 and HCT 116 were detected by qRT-PCR. (3) The down-regulated Wnt7A expression vector was constructed, and the down-regulated Wnt7A expression cell line was established. The regeneration ability of cancer cells was detected by stem cell ball formation assay, and the influence of plate cloning assay on the proliferation ability of colorectal cancer cells was detected.
    Results: (1) The positive rates of Wnt7a in normal colorectal mucosa, colorectal adenoma and colorectal adenocarcinoma tissues gradually increased,Wnt7a are closely related to the degree of colorectal adenocarcinoma differentiation, lymph node metastasis and Duke stage. (2) The expression level of Wnt7a in colorectal cancer cells was higher than that in normal colorectal epithelial cells. (3) The down-regulation of Wnt7A reduced the proliferation ability of colorectal cancer cells.
    Conclusions: Wnt7a promotes the occurrence and development of colorectal adenocarcinoma.
    Keywords:  Wnt7a; colorectal cancer; development; occurrence; proliferation
  30. Int J Mol Sci. 2021 Mar 27. pii: 3464. [Epub ahead of print]22(7):
      The phospatidylinositol-3 kinase (PI3K) pathway is a crucial intracellular signaling pathway which is mutated or amplified in a wide variety of cancers including breast, gastric, ovarian, colorectal, prostate, glioblastoma and endometrial cancers. PI3K signaling plays an important role in cancer cell survival, angiogenesis and metastasis, making it a promising therapeutic target. There are several ongoing and completed clinical trials involving PI3K inhibitors (pan, isoform-specific and dual PI3K/mTOR) with the goal to find efficient PI3K inhibitors that could overcome resistance to current therapies. This review focuses on the current landscape of various PI3K inhibitors either as monotherapy or in combination therapies and the treatment outcomes involved in various phases of clinical trials in different cancer types. There is a discussion of the drug-related toxicities, challenges associated with these PI3K inhibitors and the adverse events leading to treatment failure. In addition, novel PI3K drugs that have potential to be translated in the clinic are highlighted.
    Keywords:  PI3K inhibitors; PIK3CA; cancer; resistance