bims-pimaco 2022-03-20 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2022–03–20
twelve papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

PLHPP2 inhibits the stemness of colorectal cancer by inactivating the Nrf2 signaling pathway.
eIF3a regulation of mTOR signaling and translational control via HuR in cellular response to DNA damage.
KRAS and NRAS mutational analysis in plasma ctDNA from patients with metastatic colorectal cancer by real-time PCR and digital PCR.
Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.
Rheb regulates nuclear mTORC1 activity independent of farnesylation.
The potential of PIK3CA, KRAS, BRAF, and APC hotspot mutations as a non-invasive detection method for colorectal cancer.
Development of a miRNA-based classifier for detection of colorectal cancer molecular subtypes.
Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt.
GNE-493 inhibits prostate cancer cell growth via Akt-mTOR-dependent and -independent mechanisms.
Identification of defined structural elements within TOR2 kinase required for TOR Complex 2 assembly and function in S. cerevisiae.
The Role of Cystine/Glutamate Antiporter SLC7A11/xCT in the Pathophysiology of Cancer.
Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake.

J Cancer. 2022 ;13(4): 1313-1323

PLHPP2 inhibits the stemness of colorectal cancer by inactivating the Nrf2 signaling pathway.

Xiong Yongfu, Zhou He, Huang Xujian, Huang Jiemei, Yang Gang, Zhixiong Lei, Li Jingdong.

  Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a critical regulator of cellular homeostasis and acts as a tumor suppressor in multiple human cancers. However, its exact biological function in colorectal cancer (CRC) and the underlying molecular mechanism remain poorly understood. The correlation between the transcription and protein abundance of PHLPP2 was analyzed using proteomic and corresponding transcriptional data. Immunohistochemistry was used to validate the protein expression and the role of PHLPP2 in patient prognosis. In addition, a series of experiments in vitro and in vivo were performed to investigate the underlying molecular mechanism. Immunohistochemical staining of a CRC tissue microarray revealed that PHLPP2 protein expression was significantly downregulated compared to that in adjacent normal tissues. Low expression of PHLPP2 was an independent prognostic risk factor for poor survival. A nomogram established by integrating PHLPP2 expression and traditional clinicopathological factors achieved more reliable prognostic assessment in CRC patients. Additionally, PHLPP2 overexpression suppressed CRC cell migration, invasion and stemness in vitro as well as tumorigenesis in vivo. Further experiments revealed that upregulation of PHLPP2 increased ROS levels by suppressing the Nrf2-ARE signaling pathway, which inhibited the stemness of CRC cells. Moreover, incubation with sulforaphane, a selective chemical agonist of Nrf2, reversed this inhibitory effect in CRC. PHLPP2 acts as a tumor suppressor gene in CRC by restraining the Nrf2-ARE signaling pathway and increasing ROS levels, affecting the stemness of CRC cells. These anticancer molecular mechanisms indicate PHLLPP2's significant clinical value in prognosis prediction and targeted therapy.

Keywords:  Nrf2; PHLPP2; colorectal cancer; stemness

DOI:  https://doi.org/10.7150/jca.65444
Oncogene. 2022 Mar 12.

eIF3a regulation of mTOR signaling and translational control via HuR in cellular response to DNA damage.

Shijie Ma, Zizheng Dong, Yanfei Huang, Jing-Yuan Liu, Jian-Ting Zhang.

eIF3a (eukaryotic translation initiation factor 3a), a subunit of the eIF3 complex, has been suggested to play a regulatory role in protein synthesis and in cellular response to DNA-damaging treatments. S6K1 is an effector and a mediator of mTOR complex 1 (mTORC1) in regulating protein synthesis and integrating diverse signals into control of cell growth and response to stress. Here, we show that eIF3a regulates S6K1 activity by inhibiting mTORC1 kinase via regulating Raptor synthesis. The regulation of Raptor synthesis is via eIF3a interaction with HuR (human antigen R) and binding of the eIF3a-HuR complex to the 5'-UTR of Raptor mRNA. Furthermore, mTORC1 may mediate eIF3a function in cellular response to cisplatin by regulating synthesis of NER proteins and NER activity. Taken together, we conclude that the mTOR signaling pathway may also be regulated by translational control and mediate eIF3a regulation of cancer cell response to cisplatin by regulating NER protein synthesis.

DOI: https://doi.org/10.1038/s41388-022-02262-5
Int J Colorectal Dis. 2022 Mar 18.

KRAS and NRAS mutational analysis in plasma ctDNA from patients with metastatic colorectal cancer by real-time PCR and digital PCR.

Manuela Pinheiro, Ana Peixoto, Patricia Rocha, Isabel Veiga, Carla Pinto, Catarina Santos, Pedro Pinto, Joana Guerra, Carla Escudeiro, Ana Barbosa, João Silva, Manuel R Teixeira.

   PURPOSE: Mutations in the KRAS and NRAS (RAS) genes are negative predictors of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC). The detection of mutations in circulating tumor DNA (ctDNA) has emerged as a less invasive strategy to assess the molecular profile of mCRC patients. We aimed to perform RAS mutational analysis in ctDNA from mCRC patients using BEAMing Digital PCR (OncoBEAM) and Idylla ctDNA qPCR and evaluate the concordance rate with RAS mutational status in tumor tissue and between these two methodologies with different limits of detection.
METHODS: Blood samples were collected from 47 mCRC patients previously tested for RAS mutations in tumor tissue. DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit, and RAS mutation analysis was conducted using OncoBEAM RAS CRC and Idylla ctRAS assays.
RESULTS: The overall agreement between tumor tissue and ctDNA analyses was 83% and 78.7% using the OncoBEAM and Idylla assays, respectively, with the concordance being 96.2% and 88.5% in naive treatment patients. The overall agreement between OncoBEAM and Idylla ctDNA analyses was 91.7%.
CONCLUSIONS: Analysis of ctDNA is a viable strategy for clinical management of mCRC patients. Although the OncoBEAM assay sensitivity is somewhat higher, the fully automated Idylla platform also has good performance, while being cheaper and much less labor-intensive, for the detection of RAS mutations in plasma, either at diagnosis or after progression when considering anti-EGFR treatment rechallenge.

Keywords:  Anti-EGFR therapy; Circulating tumor DNA; Metastatic colorectal cancer; RAS mutation; Tumor tissue testing

DOI:  https://doi.org/10.1007/s00384-022-04126-6
FEBS Open Bio. 2022 Mar 18.

Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.

Oro Uchenunu, Alexander V Zhdanov, Phillipe Hutton, Predrag Jovanovic, Ye Wang, Dmitry E Andreev, Laura Hulea, David J Papadopoli, Daina Avizonis, Pavel V Baranov, Michael N Pollak, Dmitri B Papkovsky, Ivan Topisirovic.

  Mutations in genes encoding cytochrome c oxidase (COX; mitochondrial complex IV) subunits and assembly factors (e.g., SCO1, SCO2, COA6) are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumour suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the IGF1R/AKT axis with paradoxical downregulation of mTOR signaling which was accompanied by increased AMPK activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

Keywords:  AKT; AMPK; SCO2; cytochrome C oxidase; mTOR; metabolism; mitochondrial dysfunction

DOI:  https://doi.org/10.1002/2211-5463.13398
Cell Chem Biol. 2022 Mar 04. pii: S2451-9456(22)00087-3. [Epub ahead of print]

Rheb regulates nuclear mTORC1 activity independent of farnesylation.

Yanghao Zhong, Xin Zhou, Kun-Liang Guan, Jin Zhang.

  The small GTPase Ras homolog enriched in brain (Rheb) plays a critical role in activating the mechanistic target of rapamycin complex 1 (mTORC1), a signaling hub that regulates various cellular functions. We recently observed nuclear mTORC1 activity, raising an intriguing question as to how Rheb, which is known to be farnesylated and localized to intracellular membranes, regulates nuclear mTORC1. In this study, we found that active Rheb is present in the nucleus and required for nuclear mTORC1 activity. We showed that inhibition of farnesyltransferase reduced cytosolic, but not nuclear, mTORC1 activity. Furthermore, a farnesylation-deficient Rheb mutant, with preferential nuclear localization and specific lysosome tethering, enables nuclear and cytosolic mTORC1 activities, respectively. These data suggest that non-farnesylated Rheb is capable of interacting with and activating mTORC1, providing mechanistic insights into the molecular functioning of Rheb as well as regulation of the recently observed, active pool of nuclear mTORC1.

Keywords:  Compartmentation; PTM; TSC; biosensor; lipid modification; mTOR; small GTPase

DOI:  https://doi.org/10.1016/j.chembiol.2022.02.006
Mol Cell Probes. 2022 Mar 13. pii: S0890-8508(22)00018-4. [Epub ahead of print] 101807

The potential of PIK3CA, KRAS, BRAF, and APC hotspot mutations as a non-invasive detection method for colorectal cancer.

Maryam Alizadeh-Sedigh, Habibollah Mahmoodzadeh, Mohammad Sadegh Fazeli, Mohammad Haddadi-Aghdam, Ladan Teimoori-Toolabi.

   BACKGROUND: Early diagnosis of colorectal cancer (CRC) can lead to prompt treatment modalities. Circulating cell-free DNA (cfDNA) analysis provides an alternative non-invasive procedure for the study of the molecular profiles of the corresponding tumor tissue. In this study, we aimed to investigate PIK3CA, KRAS, BRAF, and APC hotspot mutations in CRC tumor tissue, besides evaluating the diagnostic performance of KRAS, BRAF, and PIK3CA mutations in the plasma cfDNA.
METHOD: Primary CRC tissue samples and paired plasma samples were collected from 70 patients. After DNA extraction, PCR-direct sequencing was used to screen for mutations in PIK3CA exon 9 and APC exon 15 in tumor tissues. Amplification Refractory Mutation System (ARMS)-quantitative PCR (qPCR) was used to evaluate KRAS codon 12 and 13, BRAF V600E, and PIK3CA exon 9 hotspot mutations.
RESULTS: PIK3CA exon 9 hotspot mutations were detected in 47.1% of tumor tissues and 20% of paired plasma cfDNA samples by ARMS-qPCR method, while Sanger sequencing did not identify any mutation in PIK3CA exon 9. The KRAS exon 2 mutations were detected in 71.4% and 34.3% of tumor tissue samples and paired plasma cfDNA respectively. BRAF V600E mutation was observed in 17.1% and 4.3% of tissue DNA and plasma cfDNA respectively. A panel of PIK3CA, KRAS, and BRAF showed a sensitivity of 61% and a specificity of 100% (AUC = 0.803). APC hotspot mutations were observed in 76.8% of CRC tissue samples. APC mutations were not analyzed in the plasma samples. The co-existence of KRAS/PIK3CA/APC gene mutations encompassed the highest frequency among all combinations of mutations. BRAF and PIK3CA mutations were significantly more frequent in older patients.
CONCLUSION: We demonstrated that a panel consisting of PIK3CA, KRAS, and BRAF mutations showed good diagnostic performance for detecting CRC in the plasma cfDNA.

Keywords:  APC, (Adenomatous polyposis coli); BRAF, (v-Raf murine sarcoma viral oncogene homolog B); Biomarker; Cell free DNA; Colorectal neoplasm; KRAS, (Kirsten rat sarcoma viral oncogene homolog); Liquid biopsy; Mutation; PIK3CA, (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha)

DOI:  https://doi.org/10.1016/j.mcp.2022.101807
Mol Oncol. 2022 Mar 17.

Development of a miRNA-based classifier for detection of colorectal cancer molecular subtypes.

Ronja S Adam, Dennis Poel, Leandro Ferreira Moreno, Joey Spronck, Tim R de Back, Arezo Torang, Patricia M Barila Gomez, Sanne Ten Hoorn, Florian Markowetz, Xin Wang, Henk M W Verheul, Tineke E Buffart, Louis Vermeulen.

  Previously, colorectal cancer (CRC) has been classified into four distinct molecular subtypes based on transcriptome data. These Consensus Molecular Subtypes (CMSs) have implications for our understanding of tumour heterogeneity and the prognosis of patients. So far, this classification has been based on the use of messenger RNAs (mRNAs), although microRNAs (miRNAs) have also been shown to play a role in tumour heterogeneity and biological differences between CMSs. In contrast to mRNAs, miRNAs have a smaller size and increased stability, facilitating their detection. Therefore, we built a miRNA-based CMS-classifier by converting the existing mRNA-based CMS-classification using machine learning (training dataset of n = 271). The performance of this miRNA-assigned CMS-classifier (CMS-miRaCl) was evaluated in several datasets, achieving an overall accuracy of ~0.72 (0.6329-0.7987) in the largest dataset (n = 158). To gain insight into the biological relevance of CMS-miRaCl, we evaluated the most important features in the classifier. We found that miRNAs previously reported to be relevant in microsatellite instable CRCs or in Wnt-signalling, were important features for CMS-miRaCl. Following further studies to validate its robustness, this miRNA-based alternative might simplify implementation of CMS-classification in clinical workflows.

Keywords:  Colorectal Cancer; Consensus Molecular Subtypes; miRNA; microRNA

DOI:  https://doi.org/10.1002/1878-0261.13210
Life Sci Alliance. 2022 Jun;pii: e202101169. [Epub ahead of print]5(6):

Single serine on TSC2 exerts biased control over mTORC1 activation mediated by ERK1/2 but not Akt.

Brittany L Dunkerly-Eyring, Shi Pan, Miguel Pinilla-Vera, Desirae McKoy, Sumita Mishra, Maria I Grajeda Martinez, Christian U Oeing, Mark J Ranek, David A Kass.

Tuberous sclerosis complex-2 (TSC2) negatively regulates mammalian target of rapamycin complex 1 (mTORC1), and its activity is reduced by protein kinase B (Akt) and extracellular response kinase (ERK1/2) phosphorylation to activate mTORC1. Serine 1364 (human) on TSC2 bidirectionally modifies mTORC1 activation by pathological growth factors or hemodynamic stress but has no impact on resting activity. We now show this modification biases to ERK1/2 but not Akt-dependent TSC2-mTORC1 activation. Endothelin-1-stimulated mTORC1 requires ERK1/2 activation and is bidirectionally modified by phospho-mimetic (S1364E) or phospho-silenced (S1364A) mutations. However, mTORC1 activation by Akt-dependent stimuli (insulin or PDGF) is unaltered by S1364 modification. Thrombin stimulates both pathways, yet only the ERK1/2 component is modulated by S1364. S1364 also has negligible impact on mTORC1 regulation by energy or nutrient status. In vivo, diet-induced obesity, diabetes, and fatty liver couple to Akt activation and are also unaltered by TSC2 S1364 mutations. This contrasts to prior reports showing a marked impact of both on pathological pressure-stress. Thus, S1364 provides ERK1/2-selective mTORC1 control and a genetic means to modify pathological versus physiological mTOR stimuli.

DOI: https://doi.org/10.26508/lsa.202101169
Cell Death Discov. 2022 Mar 16. 8(1): 120

GNE-493 inhibits prostate cancer cell growth via Akt-mTOR-dependent and -independent mechanisms.

Lu Jin, Wei Zhang, Ming-Yu Yao, Ye Tian, Bo-Xin Xue, Wei Tao.

GNE-493 is a novel PI3K/mTOR dual inhibitor with improved metabolic stability, oral bioavailability, and excellent pharmacokinetic parameters. Here GNE-493 potently inhibited viability, proliferation, and migration in different primary and established (LNCaP and PC-3 lines) prostate cancer cells, and provoking apoptosis. GNE-493 blocked Akt-mTOR activation in primary human prostate cancer cells. A constitutively-active mutant Akt1 restored Akt-mTOR activation but only partially ameliorated GNE-493-induced prostate cancer cell death. Moreover, GNE-493 was still cytotoxic in Akt1/2-silenced primary prostate cancer cells. Significant oxidative stress and programmed necrosis cascade activation were detected in GNE-493-treated prostate cancer cells. Moreover, GNE-493 downregulated Sphingosine Kinase 1 (SphK1), causing ceramide accumulation in primary prostate cancer cells. Daily single dose GNE-493 oral administration robustly inhibited the growth of the prostate cancer xenograft in the nude mice. Akt-mTOR inactivation, SphK1 downregulation, ceramide level increase, and oxidative injury were detected in GNE-493-treated prostate cancer xenograft tissues. Together, GNE-493 inhibited prostate cancer cell growth possibly through the Akt-mTOR-dependent and -independent mechanisms.

DOI: https://doi.org/10.1038/s41420-022-00911-y
Mol Biol Cell. 2022 Mar 16. mbcE21120611

Identification of defined structural elements within TOR2 kinase required for TOR Complex 2 assembly and function in S. cerevisiae.

Jennifer Tsverov, Kristina Yegorov, Ted Powers.

mTOR is a large protein kinase that assembles into two multi-subunit protein complexes, mTORC1 and mTORC2, to regulate cell growth in eukaryotic cells. While significant progress has been made in our understanding of the composition and structure of these complexes, important questions remain regarding the role of specific sequences within mTOR important for complex formation and activity. To address these issues, we have used a molecular genetic approach to explore TOR Complex assembly in budding yeast, where two closely related TOR paralogs, TOR1 and TOR2, partition preferentially into TORC1 versus TORC2, respectively. We previously identified a ∼500 amino acid segment within the N-terminal half of each protein, termed the Major Assembly Specificity (MAS) Domain, which can govern specificity in formation of each complex. In this study, we have extended the use of chimeric TOR1-TOR2 genes as a "sensitized" genetic system to identify specific subdomains rendered essential for TORC2 function, using synthetic lethal interaction analyses. Our findings reveal important design principles underlying the dimeric assembly of TORC2, as well as identify specific segments within the MAS domain critical for TORC2 function, to a level approaching single amino acid resolution. Together these findings highlight the complex and cooperative nature of TOR Complex assembly and function.

DOI: https://doi.org/10.1091/mbc.E21-12-0611
Front Oncol. 2022 ;12 858462

The Role of Cystine/Glutamate Antiporter SLC7A11/xCT in the Pathophysiology of Cancer.

Nidhi Jyotsana, Kenny T Ta, Kathleen E DelGiorno.

  SLC7A11/xCT is an antiporter that mediates the uptake of extracellular cystine in exchange for glutamate. Cystine is reduced to cysteine, which is a rate-limiting precursor in glutathione synthesis; a process that protects cells from oxidative stress and is, therefore, critical to cell growth, proliferation, and metabolism. SLC7A11 is expressed in different tissues and plays diverse functional roles in the pathophysiology of various diseases, including cancer, by regulating the processes of redox homeostasis, metabolic flexibility/nutrient dependency, immune system function, and ferroptosis. SLC7A11 expression is associated with poor prognosis and drug resistance in cancer and, therefore, represents an important therapeutic target. In this review, we discuss the molecular functions of SLC7A11 in normal versus diseased tissues, with a special focus on how it regulates gastrointestinal cancers. Further, we summarize current therapeutic strategies targeting SLC7A11 as well as novel avenues for treatment.

Keywords:  Cancer therapy; SLC7A11 (xCT); cysteine (Cys); ferroptosis; gastrointestinal tract; metabolism; oxidative stress

DOI:  https://doi.org/10.3389/fonc.2022.858462
Cell Rep. 2022 Mar 15. pii: S2211-1247(22)00245-5. [Epub ahead of print]38(11): 110509

Methylene-bridge tryptophan fatty acylation regulates PI3K-AKT signaling and glucose uptake.

Song-Hua Hu, Xia-Di He, Ji Nie, Jun-Li Hou, Jiang Wu, Xiao-Yan Liu, Yun Wei, Hui-Ru Tang, Wen-Xing Sun, Shu-Xian Zhou, Yi-Yuan Yuan, Yan-Peng An, Guo-Quan Yan, Yan Lin, Peng-Cheng Lin, Jean J Zhao, Ming-Liang Ye, Jian-Yuan Zhao, Wei Xu, Shi-Min Zhao.

  Protein fatty acylation regulates numerous cell signaling pathways. Polyunsaturated fatty acids (PUFAs) exert a plethora of physiological effects, including cell signaling regulation, with underlying mechanisms to be fully understood. Herein, we report that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) regulate PI3K-AKT signaling by modifying PDK1 and AKT2. DHA-administered mice exhibit altered phosphorylation of proteins in signaling pathways. Methylene bridge-containing DHA/EPA acylate δ1 carbon of tryptophan 448/543 in PDK1 and tryptophan 414 in AKT2 via free radical pathway, recruit both the proteins to the cytoplasmic membrane, and activate PI3K signaling and glucose uptake in a tryptophan acylation-dependent but insulin-independent manner in cultured cells and in mice. DHA/EPA deplete cytosolic PDK1 and AKT2 and induce insulin resistance. Akt2 knockout in mice abrogates DHA/EPA-induced PI3K-AKT signaling. Our results identify PUFA's methylene bridge tryptophan acylation, a protein fatty acylation that regulates cell signaling and may underlie multifaceted effects of methylene-bridge-containing PUFAs.

Keywords:  Akt signaling; DHA/EPA; fatty acylation

DOI:  https://doi.org/10.1016/j.celrep.2022.110509