bims-pimaco 2021-01-17 papers

on PI3K and MAPK signalling in colorectal cancer

Issue of 2021–01–17
eleven papers selected by
Lucas B. Zeiger, CRUK Scotland Institute, Beatson Institute for Cancer Research

Translation initiation in cancer at a glance.
The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint.
The interplay of ROS and the PI3K/Akt pathway in autophagy regulation.
Regulation of SIRT2 by Wnt/β-catenin signaling pathway in colorectal cancer cells.
PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells.
Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer.
Preoperative detection of KRAS mutated circulating tumor DNA is an independent risk factor for recurrence in colorectal cancer.
A Novel Therapeutic Approach for Colorectal Cancer Stem Cells: Blocking the PI3K/Akt Signaling Axis With Caffeic Acid.
Intracellular Porphyromonas gingivalis Promotes the Proliferation of Colorectal Cancer Cells via the MAPK/ERK Signaling Pathway.
Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells.
Radiation-Activated PI3K/AKT Pathway Promotes the Induction of Cancer Stem-Like Cells via the Upregulation of SOX2 in Colorectal Cancer.

J Cell Sci. 2021 Jan 13. pii: jcs248476. [Epub ahead of print]134(1):

Translation initiation in cancer at a glance.

Rachael C L Smith, Georgios Kanellos, Nikola Vlahov, Constantinos Alexandrou, Anne E Willis, John R P Knight, Owen J Sansom.

  Cell division, differentiation and function are largely dependent on accurate proteome composition and regulated gene expression. To control this, protein synthesis is an intricate process governed by upstream signalling pathways. Eukaryotic translation is a multistep process and can be separated into four distinct phases: initiation, elongation, termination and recycling of ribosomal subunits. Translation initiation, the focus of this article, is highly regulated to control the activity and/or function of eukaryotic initiation factors (eIFs) and permit recruitment of mRNAs to the ribosomes. In this Cell Science at a Glance and accompanying poster, we outline the mechanisms by which tumour cells alter the process of translation initiation and discuss how this benefits tumour formation, proliferation and metastasis.

Keywords:  Cancer; Cell signalling; Translation

DOI:  https://doi.org/10.1242/jcs.248476
Mol Cell Proteomics. 2020 Sep;pii: S1535-9476(20)35096-9. [Epub ahead of print]19(9): 1450-1467

The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint.

Sandhya Manohar, Qing Yu, Steven P Gygi, Randall W King.

  Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G1. Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.

Keywords:  Anaphase-promoting complex/cyclosome; G1; anaphase promoting complex (APC/C); cell cycle; cell division; enzyme inhibition; insulin signaling pathway; mitosis; ubiquitin

DOI:  https://doi.org/10.1074/mcp.RA120.002069
Biotechnol Appl Biochem. 2021 Jan 14.

The interplay of ROS and the PI3K/Akt pathway in autophagy regulation.

Dr Lakhan Kma, Dr Taranga Jyoti Baruah.

  Autophagy causes the breakdown of damaged proteins and organelles to their constituent components. The Phosphatidylinositol 3-kinase (PI3K) pathway played an important role in regulating the autophagic response of cells in reply to changing reactive oxygen species (ROS) levels. The PI3K α catalytic subunit inhibits autophagy while the β catalytic subunit promotes autophagy in response to changes in ROS levels. The downstream Akt protein acts against autophagy initiation in response to increases in ROS levels under nutrient-rich conditions. Akt acts by activating mechanistic target of rapamycin complex 1 (mTORC1) and by arresting autophagic gene expression. AMP-activated protein kinase (AMPK) protein counteracts the Akt actions. mTORC1 and mTORC2 inhibit autophagy under moderate ROS levels, but under high ROS levels, mTORC2 can promote cellular senescence via autophagy. Phosphatase and Tensin Homologue (PTEN) protein is the negative regulator of the PI3K pathway, and it has pro-autophagic activities. Studies conducted on cells treated with flavonoids and ionizing radiation showed that the moderate increase in ROS levels in the flavonoid treated groups corresponded with higher PTEN levels and lowered Akt levels leading to a higher occurrence of autophagy. In contrast, higher ROS level evoked by ionizing radiation caused a lowering of the incidence of autophagy. This article is protected by copyright. All rights reserved.

Keywords:  AMPK; Akt; Autophagy; Beclin-1; PI3K; ROS; apoptosis; cancer; flavonoid; mTOR

DOI:  https://doi.org/10.1002/bab.2104
Biochim Biophys Acta Mol Cell Res. 2021 Jan 12. pii: S0167-4889(21)00020-3. [Epub ahead of print] 118966

Regulation of SIRT2 by Wnt/β-catenin signaling pathway in colorectal cancer cells.

Chang Li, Yuning Zhou, Ji Tae Kim, Tomoko Sengoku, Michael C Alstott, Heidi L Weiss, Qingding Wang, B Mark Evers.

  Activation of the Wnt/□-catenin pathway is one of the hallmarks of colorectal cancer (CRC). Sirtuin 2 (SIRT2) protein has been shown to inhibit CRC proliferation. Previously, we reported that SIRT2 plays an important role in the maintenance of normal intestinal cell homeostasis. Here, we show that SIRT2 is a direct target gene of Wnt/□-catenin signaling in CRC cells. Inhibition or knockdown of Wnt/□-catenin increased SIRT2 promoter activity and mRNA and protein expression, whereas activation of Wnt/□-catenin decreased SIRT2 promoter activity and expression. □-catenin was recruited to the promoter of SIRT2 and transcriptionally regulated SIRT2 expression. Wnt/□-catenin inhibition increased mitochondrial oxidative phosphorylation (OXPHOS) and CRC cell differentiation. Moreover, inhibition of OXPHOS attenuated the differentiation of CRC cells induced by Wnt/□-catenin inhibition. In contrast, inhibition or knockdown of SIRT2 decreased, while overexpression of SIRT2 increased, OXPHOS activity and differentiation in CRC cells. Consistently, inhibition or knockdown or SIRT2 attenuated the differentiation induced by Wnt/□-catenin inhibition. These results demonstrate that SIRT2 is a novel target gene of the Wnt/□-catenin signaling and contributes to the differentiation of CRC cells.

Keywords:  Cell differentiation; Cell signaling; Protein expression

DOI:  https://doi.org/10.1016/j.bbamcr.2021.118966
Gut. 2021 Jan 12. pii: gutjnl-2020-323553. [Epub ahead of print]

PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells.

Laura Rosa Mangiapane, Annalisa Nicotra, Alice Turdo, Miriam Gaggianesi, Paola Bianca, Simone Di Franco, Davide Stefano Sardina, Veronica Veschi, Michele Signore, Sven Beyes, Luca Fagnocchi, Micol Eleonora Fiori, Maria Rita Bongiorno, Melania Lo Iacono, Irene Pillitteri, Gloria Ganduscio, Gaspare Gulotta, Jan Paul Medema, Alessio Zippo, Matilde Todaro, Ruggero De Maria, Giorgio Stassi.

   OBJECTIVE: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy.
DESIGN: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance.
RESULTS: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy.
CONCLUSIONS: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

Keywords:  antibody targeted therapy; colorectal cancer; drug resistance; stem cells

DOI:  https://doi.org/10.1136/gutjnl-2020-323553
Molecules. 2021 Jan 11. pii: E334. [Epub ahead of print]26(2):

Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer.

Ashraf N Abdalla, Waleed H Malki, Amal Qattan, Imran Shahid, Mohammad Akbar Hossain, Muhammad Ahmed.

  Colorectal cancer (CRC) remains one of the main causes of death worldwide and in Saudi Arabia. The toxicity and the development of resistance against 5 fluorouracil 5FU pose increasing therapeutic difficulties, which necessitates the development of personalized drugs and drug combinations.
OBJECTIVES: First, to determine the most important kinases and kinase pathways, and the amount of ABC transporters and KRAS in samples taken from Saudi CRC patients. Second, to investigate the chemosensitizing effect of LY294002 and HAA2020 and their combinations with 5FU on HT29, HT29-5FU, HCT116, and HCT116-5FU CRC cells, their effect on the three ABC transporters, cell cycle, and apoptosis, in light of the important kinase pathways resulting from the first part of this study.
METHODS: The PamChip® peptide micro-array profiling was used to determine the level of kinase and targets in the Saudi CRC samples. Next, RT-PCR, MTT cytotoxicity, Western blotting, perturbation of cell cycle, annexin V, and immunofluorescence assays were used to investigate the effect on CRC, MRC5, and HUVEC cells.
RESULTS: The kinase activity profiling highlighted the importance of the PI3K/AKT, MAPK, and the growth factors pathways in the Saudi CRC samples. PIK3CA was the most overexpressed, and it was associated with increased level of mutated KRAS and the three ABC transporters, especially ABCC1 in the Saudi samples. Next, combining HAA2020 with 5FU exhibited the best synergistic and resistance-reversal effect in the four CRC cells, and the highest selectivity indices compared to MRC5 and HUVEC normal cells. Additionally, HAA2020 with 5FU exerted significant inhibition of ABCC1 in the four CRC cells, and inhibition of PIK3CA/AKT/MAPK7/ERK in HT29 and HT29-5FU cells. The combination also inhibited EGFR, increased the preG1/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G1 phase, p21/p27, and apoptosis in HT29-5FU cells.
CONCLUSION: We have combined the PamChip kinase profiling of Saudi CRC samples with in vitro drug combination studies in four CRC cells, highlighting the importance of targeting PIK3CA and ABCC1 for Saudi CRC patients, especially given that the overexpression of PIK3CA mutations was previously linked with the lack of activity for the anti-EGFRs as first line treatment for CRC patients. The combination of HAA2020 and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.

Keywords:  5FU; ABCC1; PIK3CA; Saudi colorectal cancer; drug resistance; kinase pathway profiling

DOI:  https://doi.org/10.3390/molecules26020334
Sci Rep. 2021 Jan 11. 11(1): 441

Preoperative detection of KRAS mutated circulating tumor DNA is an independent risk factor for recurrence in colorectal cancer.

Yuki Nakamura, Shozo Yokoyama, Kenji Matsuda, Koichi Tamura, Yasuyuki Mitani, Hiromitsu Iwamoto, Yuki Mizumoto, Daisuke Murakami, Yuji Kitahata, Hiroki Yamaue.

Preoperative ctDNA status in relation to recurrence in cases of CRC remains unclear. We examined preoperative ctDNA detection by targeting KRAS gene mutations as a predictive marker for recurrence after CRC surgery. We measured the preoperative KRAS mutated ctDNA status and analyzed the correlation with clinicopathologic features of 180 patients that underwent surgery for CRC. We studied the association between preoperative KRAS mutated ctDNA and postoperative recurrence in patients (n = 150) that underwent radical surgery. KRAS mutated ctDNA was detected in 59 patients (32.8%). Median mutant allele frequency of KRAS in ctDNA was 0.20%. KRAS status in ctDNA and lymph node metastasis and distant metastasis were not significantly different. Among patients that underwent radical resection, recurrence occurred in 21 (14.0%, median follow-up 24 months). In Kaplan-Meier analysis, preoperative detection of KRAS mutated ctDNA was associated with inferior recurrence-free interval (RFI) (p = 0.002) and recurrence-free survival (RFS) (p = 0.025). In a multivariate Cox proportional hazards model, preoperative detection of KRAS mutated ctDNA was an independent factor related to both RFI (HR = 3.08; p = 0.012) and RFS (HR = 2.18; p = 0.044). Preoperative measurement of KRAS mutated ctDNA could be useful to decide postoperative treatment.

DOI: https://doi.org/10.1038/s41598-020-79909-4
Front Cell Dev Biol. 2020 ;8 585987

A Novel Therapeutic Approach for Colorectal Cancer Stem Cells: Blocking the PI3K/Akt Signaling Axis With Caffeic Acid.

Se-Ra Park, Soo-Rim Kim, In-Sun Hong, Hwa-Yong Lee.

  Cancer stem cells (CSCs) have been identified in a multiple of cancer types and resistant to traditional cancer therapies such as chemotherapeutic agents and radiotherapy, which may destroy bulk tumor cells but not all CSCs, contributing to reformation tumor masses and subsequent relapse. Moreover, it is very difficult to effectively identify and eliminate CSCs because they share some common phenotypic and functional characteristics of normal stem cells. Therefore, finding better therapeutic strategies to selectively target CSCs might be helpful to reduce subsequent malignancies. In the present study, we found that caffeic acid effectively suppresses self-renewal capacity, stem-like characteristics, and migratory capacity of CD44+ and CD133+ colorectal CSCs in vitro and in vivo. In addition, we also revealed that PI3K/Akt signaling may be linked to multiple colorectal CSC-associated characteristics, such as radio-resistance, stem-like property, and tumorigenic potential. To the best of our knowledge, this is the first study demonstrating that caffeic acid effectively targets colorectal CSC populations by inhibiting the growth and/or self-renewal capacity of colorectal CSCs through PI3K/Akt signaling in vitro and in vivo.

Keywords:  AKT signaling; caffeic acid; cancer stem cell; colorectal cancer; stem-like property

DOI:  https://doi.org/10.3389/fcell.2020.585987
Front Cell Infect Microbiol. 2020 ;10 584798

Intracellular Porphyromonas gingivalis Promotes the Proliferation of Colorectal Cancer Cells via the MAPK/ERK Signaling Pathway.

Wenxin Mu, Yiqun Jia, Xiaobing Chen, Haoyu Li, Zhi Wang, Bin Cheng.

  Porphyromonas gingivalis (P. gingivalis) is a keystone pathogen in periodontitis. However, several clinical studies have revealed an enrichment of P. gingivalis in the stool samples and colorectal mucosa of colorectal cancer patients. Thus, the goal of this study was to determine whether P. gingivalis can promote colorectal cancer progression in vitro. We established an acute infection model (24 h, multiplicity of infection =100) of P. gingivalis invasion of colorectal cancer cells to study the alterations induced by P. gingivalis in the proliferation and cell cycle of colorectal cancer cells. We observed that P. gingivalis can adhere and invade host cells a few hours after infection. Once invaded, P. gingivalis significantly promoted colorectal cancer cell proliferation, and the percentage of S phase cells was increased in the cell cycle assay. However, KDP136, a gingipain-deficient mutant of P. gingivalis 33277, showed a decreased ability to promote colorectal cancer cell proliferation, indicating that gingipain is associated with colorectal cancer cell proliferation. Furthermore, we extracted RNA from colorectal cancer cells for high-throughput sequencing analysis and reconfirmed the results by quantitative polymerase chain reaction and western blot analyses. The results suggested that the MAPK/ERK signaling pathway is significantly activated by P. gingivalis, while these changes were not observed for KDP136. In conclusion, P. gingivalis can invade cells and promote the proliferation of colorectal cancer cells by activating the MAPK/ERK signaling pathway. Gingipain is an essential virulence factor in this interaction.

Keywords:  Porphyromonas gingivalis; cell cycle; cell proliferation; colorectal neoplasms; gingipain cysteine endopeptidases

DOI:  https://doi.org/10.3389/fcimb.2020.584798
Oncogenesis. 2021 Jan 05. 10(1): 8

Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells.

Jian Wang, Xiaoye Lv, Xiutian Guo, Yanbo Dong, Peipei Peng, Fang Huang, Peng Wang, Haoqian Zhang, Jianguang Zhou, Youliang Wang, Bo Wei, Zeng-Fu Shang, Shanhu Li.

The PI3K/AKT/mTOR signaling pathway is constitutively active in PTEN-deficient cancer cells, and its targeted inhibition has significant anti-tumor effects. However, the efficacy of targeted therapies is often limited due to drug resistance. The relevant signaling pathways in PTEN-deficient cancer cells treated with the PI3K/mTOR inhibitor BEZ235 were screened using a phosphokinase array, and further validated following treatment with multiple PI3K/AKT/mTOR inhibitors or AKT knockdown. The correlation between PTEN expression levels and STAT3 kinase phosphorylation in the tissue microarrays of gastric cancer patients was analyzed by immunohistochemistry. Cell proliferation and clonogenic assays were performed on the suitably treated PTEN-deficient cancer cells. Cytokine arrays, small molecule inhibition and knockdown assays were performed to identify related factors. PTEN-deficient tumor xenografts were established in nude mice that were treated with PI3K/AKT/mTOR and/or STAT3 inhibitors. PTEN deficiency was positively correlated with low STAT3 activity. PI3K/mTOR inhibitors increased the expression and secretion of macrophage migration inhibitory factor (MIF) and activated the JAK1/STAT3 signaling pathway. Both cancer cells and in vivo tumor xenografts showed that the combined inhibition of PI3K/AKT/mTOR and STAT3 activity enhanced the inhibitory effect of BEZ235 on the proliferation of PTEN-deficient cancer cells. Our findings provide a scientific basis for a novel treatment strategy in cancer patients with PTEN deficiency.

DOI: https://doi.org/10.1038/s41389-020-00292-w
Cells. 2021 Jan 12. pii: E135. [Epub ahead of print]10(1):

Radiation-Activated PI3K/AKT Pathway Promotes the Induction of Cancer Stem-Like Cells via the Upregulation of SOX2 in Colorectal Cancer.

Ji-Hye Park, Young-Heon Kim, Sehwan Shim, Areumnuri Kim, Hyosun Jang, Su-Jae Lee, Sunhoo Park, Songwon Seo, Won Il Jang, Seung Bum Lee, Min-Jung Kim.

  The current treatment strategy for patients with aggressive colorectal cancer has been hampered by resistance to radiotherapy and chemotherapy due to the existence of cancer stem-like cells (CSCs). Recent studies have shown that SOX2 expression plays an important role in the maintenance of CSC properties in colorectal cancer. In this study, we investigated the induction and regulatory role of SOX2 following the irradiation of radioresistant and radiosensitive colorectal cancer cells. We used FACS and western blotting to analyze SOX2 expression in cells. Among the markers of colorectal CSCs, the expression of CD44 increased upon irradiation in radioresistant cells. Further analysis revealed the retention of CSC properties with an upregulation of SOX2 as shown by enhanced resistance to radiation and metastatic potential in vitro. Interestingly, both the knockdown and overexpression of SOX2 led to increase in CD44+ population and induction of CSC properties in colorectal cancer following irradiation. Furthermore, selective genetic and pharmacological inhibition of the PI3K/AKT pathway, but not the MAPK pathway, attenuated SOX2-dependent CD44 expression and metastatic potential upon irradiation in vitro. Our findings suggested that SOX2 regulated by radiation-induced activation of PI3K/AKT pathway contributes to the induction of colorectal CSCs, thereby highlighting its potential as a therapeutic target.

Keywords:  PI3K/AKT; SOX2; cancer-stem like cells; colorectal cancer; radioresistance

DOI:  https://doi.org/10.3390/cells10010135