bims-instec 2022-05-08 papers

bims-instec

Biomed News

on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration

Issue of 2022–05–08
twenty-two papers selected by
Maria-Virginia Giolito, IRFAC/UMR-S1113 INSERM

PDGFRα-induced stromal maturation is required to restrain postnatal intestinal epithelial stemness and promote defense mechanisms.
Disulfiram Protects Against Radiation-Induced Intestinal Injury in Mice.
Organoids as a tool for understanding immune-mediated intestinal regeneration and development.
Differential effects of 5-fluorouracil and oxaliplatin on autophagy in chemoresistant colorectal cancer cells.
Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells.
Single-cell transcriptome highlights a multilayer regulatory network on an invasive trajectory within colorectal cancer progression.
UHMK1 aids colorectal cancer cell proliferation and chemoresistance through augmenting IL-6/STAT3 signaling.
Drug resistance in cancer therapy: the Pandora's Box of cancer stem cells.
Targeting DCLK1 overcomes 5-fluorouracil resistance in colorectal cancer through inhibiting CCAR1/β-catenin pathway-mediated cancer stemness.
MTG16 (CBFA2T3) regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors.
Chemotherapy-induced Reversion of Mutant RAS to Wild-type RAS in Metastatic Colorectal Cancer.
Temozolomide treatment alters mismatch repair and boosts mutational burden in tumor and blood of colorectal cancer patients.
The Evolving Treatment Landscape in BRAF-V600E-Mutated Metastatic Colorectal Cancer.
DNA Repair-Related Gene Signature in Predicting Prognosis of Colorectal Cancer Patients.
BET inhibitor JQ1 enhances anti-tumor immunity and synergizes with PD-1 blockade in CRC.
Cancer Cells Induce Delayed DNA Breaks to Resist Radiation Therapy.
Cell-free circulating tumor DNA in colorectal cancer: a proof of concept with simplified methodology.
Effectiveness of Hyperthermia as Monotherapy and Adjuvant Therapy Approaches Against an In Vitro Model of Colorectal Carcinoma.
Orthotopic Xenografts of Colorectal Cancer Stem Cells.
Genomic Landscape of Primary Tumor Site and Clinical Outcome for Patients with Metastatic Colorectal Cancer Receiving Standard-of-Care Chemotherapy.
Oxaliplatin induces ferroptosis and oxidative stress in HT29 colorectal cancer cells by inhibiting the Nrf2 signaling pathway.
The origins of cancer cell dormancy.

Cell Stem Cell. 2022 May 05. pii: S1934-5909(22)00158-8. [Epub ahead of print]29(5): 856-868.e5

PDGFRα-induced stromal maturation is required to restrain postnatal intestinal epithelial stemness and promote defense mechanisms.

Jean-Marie Jacob, Selene E Di Carlo, Igor Stzepourginski, Anthony Lepelletier, Papa Diogop Ndiaye, Hugo Varet, Rachel Legendre, Etienne Kornobis, Adam Benabid, Giulia Nigro, Lucie Peduto.

  After birth, the intestine undergoes major changes to shift from an immature proliferative state to a functional intestinal barrier. By combining inducible lineage tracing and transcriptomics in mouse models, we identify a prodifferentiation PDGFRαHigh intestinal stromal lineage originating from postnatal LTβR+ perivascular stromal progenitors. The genetic blockage of this lineage increased the intestinal stem cell pool while decreasing epithelial and immune maturation at weaning age, leading to reduced postnatal growth and dysregulated repair responses. Ablating PDGFRα in the LTBR stromal lineage demonstrates that PDGFRα has a major impact on the lineage fate and function, inducing a transcriptomic switch from prostemness genes, such as Rspo3 and Grem1, to prodifferentiation factors, including BMPs, retinoic acid, and laminins, and on spatial organization within the crypt-villus and repair responses. Our results show that the PDGFRα-induced transcriptomic switch in intestinal stromal cells is required in the first weeks after birth to coordinate postnatal intestinal maturation and function.

Keywords:  IESC; Lymphotoxin beta receptor; epithelial differentiation; inflammation; intestinal barrier; intestinal epithelial stem cells; intestinal repair; perivascular; postnatal intestinal maturation; rsubepithelial fibroblasts; stromal niches

DOI:  https://doi.org/10.1016/j.stem.2022.04.005
Front Pharmacol. 2022 ;13 852669

Disulfiram Protects Against Radiation-Induced Intestinal Injury in Mice.

Qingwen Yuan, Renjun Peng, Huijie Yu, Sinian Wang, Zhongmin Chen, Suhe Dong, Wei Li, Bo Cheng, Qisheng Jiang, Yuwen Cong, Fengsheng Li, Changzheng Li.

  Radiation-induced intestinal injury (RIII) occurs after high doses of radiation exposure. RIII restricts the therapeutic efficacy of radiotherapy in cancer and increases morbidity and mortality in nuclear disasters. Currently, there is no approved agent for the prevention or treatment of RIII. Here, we reported that the disulfiram, an FDA-approved alcohol deterrent, prolonged the survival in mice after lethal irradiation. Pretreatment with disulfiram inhibited proliferation within 24 h after irradiation, but improved crypt regeneration at 3.5 days post-irradiation. Mechanistically, disulfiram promoted Lgr5+ intestinal stem cells (ISCs) survival and maintained their ability to regenerate intestinal epithelium after radiation. Moreover, disulfiram suppresses DNA damage accumulation, thus inhibits aberrant mitosis after radiation. Unexpectedly, disulfiram treatment did not inhibit crypt cell apoptosis 4 h after radiation and the regeneration of crypts from PUMA-deficient mice after irradiation was also promoted by disulfiram. In conclusion, our findings demonstrate that disulfiram regulates the DNA damage response and survival of ISCs through affecting the cell cycle. Given its radioprotective efficacy and decades of application in humans, disulfiram is a promising candidate to prevent RIII in cancer therapy and nuclear accident.

Keywords:  DNA damage; Lgr5+ stem cell; disulfiram; radiation-induced intestinal injury; radioprotector

DOI:  https://doi.org/10.3389/fphar.2022.852669
Development. 2022 Apr 15. pii: dev199904. [Epub ahead of print]149(8):

Organoids as a tool for understanding immune-mediated intestinal regeneration and development.

Geraldine M Jowett, Isabelle Coales, Joana F Neves.

  The traditional view of immune cells is that their role within the body is to combat infections; however, it is becoming increasingly clear that they also perform tasks that are not classically associated with inflammation and pathogen clearance. These functions are executed deep within tissues, which are often poorly accessible and subject to environmental variability, especially in humans. Here, we discuss how multicellular 3D systems in a dish - organoids - are transitioning from a proof-of-principle approach to a timely, robust and reliable tool. Although we primarily focus on recent findings enabled by intestinal organoids co-cultured with lymphocytes, we posit that organoid co-culture systems will support future efforts to disentangle the interactions between a plethora of different cell types throughout development, homeostasis, regeneration and disease.

Keywords:  Immune cells; Innate lymphoid cells; Intestine; Organoids

DOI:  https://doi.org/10.1242/dev.199904
J Cell Biochem. 2022 May 01.

Differential effects of 5-fluorouracil and oxaliplatin on autophagy in chemoresistant colorectal cancer cells.

Vilmante Zitkute, Egle Kukcinaviciute, Violeta Jonusiene, Vytaute Starkuviene, Ausra Sasnauskiene.

  Macroautophagy (hereafter autophagy) is one of the adaptive pathways that contribute to cancer cell chemoresistance. Despite the fact that autophagy can both promote and inhibit cell death, there is mounting evidence that in the context of anticancer treatment, it predominantly functions as a cell survival mechanism. Therefore, silencing of key autophagy genes emerges as a potent strategy to reduce chemoresistance. Though the importance of autophagy in chemoresistance is established, the changes in autophagy in the case of acquired chemoresistance are poorly understood. In this study, we aimed to determine the changes of autophagy in the cellular model of acquired chemoresistance of colorectal cancer cell lines HCT116 and SW620, induced by 5-fluorouracil (5-FU) or oxaliplatin (OxaPt) treatment, and determine the susceptible factors for autophagy inhibition. Our results demonstrate that in the context of autophagy, 5-FU and OxaPt have different effects on HCT116 and SW620 cell lines and their chemoresistant sublines. 5-FU inhibits autophagic flux, while changes in the flux after OxaPt treatment are cell type- and dose-dependent, inducing autophagy reduction or increase. The chemoresistant subline of HCT116 cells derived by OxaPt differs from the subline derived by 5-FU treatment - it responds to OxaPt by upregulating ATG7 protein level and autophagic flux, in contrast to downregulation in cells derived by 5-FU. Moreover, 5-FU and OxaPt treatments significantly modulate protein levels of core-autophagy proteins ATG7 and ATG12. The potential effects of 5-FU and OxaPt on ATG protein levels should be taken into account to reduce chemoresistance by applying small interferingRNAs, targeting ATG proteins.

Keywords:  5-fluorouracil; ATG12; ATG7; autophagy; chemoresistance; colorectal cancer; oxaliplatin

DOI:  https://doi.org/10.1002/jcb.30267
Stem Cell Reports. 2022 Apr 25. pii: S2213-6711(22)00194-1. [Epub ahead of print]

Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells.

Keith A Breau, Meryem T Ok, Ismael Gomez-Martinez, Joseph Burclaff, Nathan P Kohn, Scott T Magness.

  Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.

Keywords:  2D monolayer cultures; CRISPR/Cas9; OLFM4; PiggyBac; electroporation; human ISC marker; microphysiological device; planar crypt-microarray; transfection; transgenic

DOI:  https://doi.org/10.1016/j.stemcr.2022.04.005
J Cancer Res Clin Oncol. 2022 May 06.

Single-cell transcriptome highlights a multilayer regulatory network on an invasive trajectory within colorectal cancer progression.

Tong Zhou, Chunhua Li.

   PURPOSE: Colorectal cancer (CRC) is one of the most common and fatal gastrointestinal malignancies, in which cancer stem cells (CSCs) were identified to enable tumor heterogeneity and initiate tumor formation. However, the process from CSCs to invasion cells is unconfirmed.
METHODS: Several bioinformatics methods, including clustering, pseudotime analysis, gene set variation analysis and gene ontology enrichment, were used to construct a path of gradual transformation of CSCs to invasive cells, called "stem-to-invasion path". A large amount of signaling interactions were collated to build the multilayer regulatory network. Kaplan-Meier curve and time-dependent ROC method were applied to reveal prognostic values.
RESULTS: We validated the heterogeneity of cells in the tumor microenvironment and revealed the presence of malignant epithelial cells with high invasive potential within primary colonic carcinomas. Next, the "stem-to-invasion path" was identified through constructing a branching trajectory with cancer cells arranged in order. A multilayer regulatory network considered as the vital factor involved in acquiring invasion characteristics underlying the path was built to elucidate the interactions between tumor cell and tumor-associated microenvironment. Then we further identified a novel combinatorial biomarker that can be used to assess the prognosis for CRC patients, and validated its predictive robustness on the independent dataset.
CONCLUSION: Our work provides new insights into the acquisition of invasive potential in primary tumor cells, as well as potential therapeutic targets for CRC invasiveness, which may be useful for the cancer research and clinical treatment.

Keywords:  Colorectal cancer; Multilayer regulatory network; Novel combinatorial biomarker; Single-cell RNA transcriptome; Stem-to-invasion path

DOI:  https://doi.org/10.1007/s00432-022-04020-2
Cell Death Dis. 2022 May 02. 13(5): 424

UHMK1 aids colorectal cancer cell proliferation and chemoresistance through augmenting IL-6/STAT3 signaling.

Xuedi Gao, Wenfang Bao, Jin Bai, Kailing Fan, Li Li, Yandong Li.

UHMK1, a serine/threonine kinase with a U2AF homology motif, is implicated in RNA processing and protein phosphorylation. Increasing evidence has indicated its involvement in tumorigenesis. However, it remains to be elucidated whether UHMK1 plays a role in the development of colorectal cancer (CRC). Here, we demonstrated that UHMK1 was frequently upregulated in CRC samples compared with adjacent normal tissue and high expression of UHMK1 predicted poor outcomes. Knockdown of UHMK1 by siRNAs restrained CRC cell proliferation and increased oxaliplatin sensitivity, whereas overexpression of UHMK1 promoted CRC cell growth and oxaliplatin resistance, suggesting that UHMK1 plays important oncogenic roles in CRC. Mechanistically, we showed that UHMK1 had a significant effect on IL6/STAT3 signaling by interacting with STAT3. The interaction of UHMK1 with STAT3 enhanced STAT3 activity in regulating gene transcription. Furthermore, we found that STAT3 could in turn transcriptionally activate UHMK1 expression in CRC cells. The complementary experiments for cell growth and oxaliplatin resistance indicated the interdependent relationship between UHMK1 and STAT3. Thus, these collective findings uncovered a new UHMK1/STAT3 positive feedback regulatory loop contributing to CRC development and chemoresistance.

DOI: https://doi.org/10.1038/s41419-022-04877-8
Stem Cell Res Ther. 2022 May 03. 13(1): 181

Drug resistance in cancer therapy: the Pandora's Box of cancer stem cells.

Hamed Rezayatmand, Mahboobeh Razmkhah, Iman Razeghian-Jahromi.

  Drug resistance is the main culprit of failure in cancer therapy that may lead to cancer relapse. This resistance mostly originates from rare, but impactful presence of cancer stem cells (CSCs). Ability to self-renewal and differentiation into heterogeneous cancer cells, and harboring morphologically and phenotypically distinct cells are prominent features of CSCs. Also, CSCs substantially contribute to metastatic dissemination. They possess several mechanisms that help them to survive even after exposure to chemotherapy drugs. Although chemotherapy is able to destroy the bulk of tumor cells, CSCs are left almost intact, and make tumor entity resistant to treatment. Eradication of a tumor mass needs complete removal of tumor cells as well as CSCs. Therefore, it is important to elucidate key features underlying drug resistance raised by CSCs in order to apply effective treatment strategies. However, the challenging point that threatens safety and specificity of chemotherapy is the common characteristics between CSCs and normal peers such as signaling pathways and markers. In the present study, we tried to present a comprehensive appraisal on CSCs, mechanisms of their drug resistance, and recent therapeutic methods targeting this type of noxious cells.

Keywords:  Cancer stem cell; Chemotherapy resistance; Drug resistance; Mechanism; Treatment resistance

DOI:  https://doi.org/10.1186/s13287-022-02856-6
Clin Transl Med. 2022 May;12(5): e743

Targeting DCLK1 overcomes 5-fluorouracil resistance in colorectal cancer through inhibiting CCAR1/β-catenin pathway-mediated cancer stemness.

Lanqing Wang, Lei Zhao, Zhenyu Lin, Dandan Yu, Min Jin, Pengfei Zhou, Jinghua Ren, Jing Cheng, Kunyu Yang, Gang Wu, Tao Zhang, Dejun Zhang.

   BACKGROUND: To date, 5-fluorouracil-based chemotherapy is very important for locally advanced or metastatic colorectal cancer (CRC). However, chemotherapy resistance results in tumor recurrence and metastasis, which is a major obstacle for treatment of CRC.
METHODS: In the current research, we establish 5-fluorouracil resistant cell lines and explore the potential targets associated with 5-fluorouracil resistance in CRC. Moreover, we perform clinical specimen research, in vitro and in vivo experiments and molecular mechanism research, to reveal the biological effects and the mechanism of DCLK1 promoting 5-fluorouracil resistance, and to clarify the potential clinical value of DCLK1 as a target of 5-fluorouracil resistance in CRC.
RESULTS: We discover that doublecortin-like kinase 1 (DCLK1), a cancer stem cell maker, is correlated with 5-fluorouracil resistance, and functionally promotes cancer stemness and 5-fluorouracil resistance in CRC. Mechanistically, we elucidate that DCLK1 interacts with cell cycle and apoptosis regulator 1 (CCAR1) through the C-terminal domain, and phosphorylates CCAR1 at the Ser343 site, which is essential for CCAR1 stabilisation. Moreover, we find that DCLK1 positively regulates β-catenin signalling via CCAR1, which is responsible for maintaining cancer stemness. Subsequently, we prove that blocking β-catenin inhibits DCLK1-mediated 5-fluorouracil resistance in CRC cells. Importantly, we demonstrate that DCLK1 inhibitor could block CCAR1/β-catenin pathway-mediated cancer stemness and consequently suppresses 5-fluorouracil resistant CRC cells in vitro and in vivo.
CONCLUSIONS: Collectively, our findings reveal that DCLK1 promotes 5-fluorouracil resistance in CRC by CCAR1/β-catenin pathway-mediated cancer stemness, and suggest that targeting DCLK1 might be a promising method to eliminate cancer stem cells for overcoming 5-fluorouracil resistance in CRC.

Keywords:  CCAR1; DCLK1; cancer stemness; chemoresistence; colorectal cancer; β-catenin

DOI:  https://doi.org/10.1002/ctm2.743
JCI Insight. 2022 May 03. pii: e153045. [Epub ahead of print]

MTG16 (CBFA2T3) regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors.

Rachel E Brown, Justin Jacobse, Shruti A Anant, Koral M Blunt, Bob Chen, Paige N Vega, Chase T Jones, Jennifer M Pilat, Frank Revetta, Aidan H Gorby, Kristy R Stengel, Yash A Choksi, Kimmo Palin, M Blanca Piazuelo, Mary K Washington, Ken S Lau, Jeremy A Goettel, Scott W Hiebert, Sarah P Short, Christopher S Williams.

  Aberrant epithelial differentiation and regeneration contribute to colon pathologies including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). MTG16 (CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium (DSS)-induced colitis. Transcriptomic analyses implicated increased E box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a novel mouse model with a point mutation that attenuates MTG16:E protein interactions (Mtg16P209T), we established that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein-mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared to unaffected controls. Finally, uncoupling MTG16:E protein interactions partially phenocopied the enhanced tumorigenicity of Mtg16-/- colon in the azoxymethane(AOM)/DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer.

Keywords:  Cell Biology; Gastroenterology; Inflammatory bowel disease

DOI:  https://doi.org/10.1172/jci.insight.153045
Anticancer Res. 2022 May;42(5): 2625-2635

Chemotherapy-induced Reversion of Mutant RAS to Wild-type RAS in Metastatic Colorectal Cancer.

Sumito Sato, Y O Mikayama, Manabu Shiozawa, Suguru Nukada, Kenta Iguchi, Hironao Okamoto, Takashi Kohmura, Keisuke Kazama, Kuniya Tanaka, Takashi Oshima, Yasushi Rino.

   BACKGROUND/AIM: Secondary mutation of mutated RAS, induced by chemotherapy, is thought to be rare. However, introduction of liquid biopsy (LB) has made it possible to monitor RAS status in patients' plasma throughout the course of chemotherapy for metastatic colorectal cancer (mCRC), and disappearance of the RAS mutation (RAS-mt), i.e., the NeoRAS-wt phenomenon, has been reported and is receiving attention, especially with respect to treatment implications.
PATIENTS AND METHODS: A prospective study of 129 patients undergoing chemotherapy for mCRC (RAS-wt, n=65; RAS-mt, n=64) was carried out. Plasma RAS status was monitored in these patients by LB. Relations between secondary genetic change, chemotherapy, and 6-month disease outcomes were analyzed. The effect of anti-EGFR mAb therapy on NeoRAS-wt mCRC was also examined.
RESULTS: NeoRAS-wt was detected in 27 (43.5%) RAS-mt patients overall and in all patients with a G12S or Q61H mutation. First-line treatment was more effective among NeoRAS-wt patients than non-NeoRAS-wt patients (70.9% vs. 48.6% overall response rate, p=0.087), and the time from treatment to LB was shorter in this group. Six-month outcomes were significantly better in the NeoRAS-wt group (p<0.001), and conversion to NeoRAS-wt was found to be predictive of a good outcome (OR=7.886, 95% CI=2.458-25.30; p<0.001). Anti-EGFR mAb therapy was found to restrict disease progression in NeoRAS-wt patients.
CONCLUSION: Conversion to NeoRAS-wt is relatively frequent, and it may predict good responses to treatment. Anti-EGFR mAb therapy was effective for our NeoRAS-wt patients. Detection of NeoRAS-wt by LB may significantly change the indication for anti-EGFR mAb therapy and the mCRC treatment strategy.

Keywords:  Colorectal cancer; anti-EGFR mAb; chemotherapy; liquid biopsy

DOI:  https://doi.org/10.21873/anticanres.15740
Cancer Discov. 2022 May 06. pii: candisc.1434.2021. [Epub ahead of print]

Temozolomide treatment alters mismatch repair and boosts mutational burden in tumor and blood of colorectal cancer patients.

Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Alessio Amatu, Marco Macagno, Ludovic Barault, Andrea Cassingena, Alice Bartolini, Paolo Luraghi, Gianluca Mauri, Paolo Battuello, Nicola Personeni, Maria Giulia Zampino, Valeria Pessei, Pietro Paolo Vitiello, Federica Tosi, Laura Idotta, Federica Morano, Emanuele Valtorta, Emanuela Bonoldi, Giovanni Germano, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli.

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, while MMR-deficient (MMRd) tumors often respond to immune-checkpoint-blockade. We previously reported that treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR-deficiency, increased tumor mutational burden (TMB) and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-Methylguanine-DNA-methyltransferase (MGMT) deficient, MMR-proficient, RAS mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A patient's subset whose tumors displayed the MSH6 mutation, the TMZ mutational signature and increased TMB, achieved disease stabilization upon pembrolizumab treatment.

DOI: https://doi.org/10.1158/2159-8290.CD-21-1434
Am Soc Clin Oncol Educ Book. 2022 Apr;42 1-10

The Evolving Treatment Landscape in BRAF-V600E-Mutated Metastatic Colorectal Cancer.

Josep Tabernero, Javier Ros, Elena Élez.

Between 8% and 12% of patients with metastatic colorectal cancer (mCRC) harbor a BRAF-V600E mutation in their tumors, which is associated with a poor response to standard chemotherapy and short overall survival. Moreover, nearly 30% of BRAF-V600E mCRC tumors also have microsatellite instability. Transcriptomic signatures suggest a strong immunogenic biologic background for most of these tumors. In contrast to the melanoma context, single-agent BRAF inhibition does not achieve clinical benefit in BRAF-V600E mCRC. Different preclinical/translational studies have elucidated that, in this context, upon BRAF inhibition, there is immediate signal upregulation via the EGFR, and therefore an anti-EGFR treatment should be added to the BRAF inhibitor. Several phase II studies have confirmed the activity of BRAF inhibitors combined with EGFR-directed monoclonal antibodies in patients with BRAF-V600E mCRC. The role of other mitogen-activated protein kinase inhibitors, such as mitogen-activated protein kinase kinase or PI3K inhibitors, remains unclear. The phase III BEACON clinical trial confirmed the BRAF/EGFR inhibitor combination of encorafenib/cetuximab as the new standard of care for BRAF-V600E mCRC after at least one previous line of systemic therapy. Novel approaches for managing BRAF-V600E mCRC include, among others, triple combinations of BRAF inhibitors and anti-EGFR antibodies combined with immune checkpoint inhibitors in the microsatellite instability population and evaluation of the encorafenib/cetuximab treatment in combination with standard chemotherapy with bevacizumab in the first-line setting.

DOI: https://doi.org/10.1200/EDBK_349561
Front Genet. 2022 ;13 872238

DNA Repair-Related Gene Signature in Predicting Prognosis of Colorectal Cancer Patients.

Min-Yi Lv, Wei Wang, Min-Er Zhong, Du Cai, Dejun Fan, Cheng-Hang Li, Wei-Bin Kou, Ze-Ping Huang, Xin Duan, Chuling Hu, Qiqi Zhu, Xiaosheng He, Feng Gao.

  Background: Increasing evidence have depicted that DNA repair-related genes (DRGs) are associated with the prognosis of colorectal cancer (CRC) patients. Thus, the aim of this study was to evaluate the impact of DNA repair-related gene signature (DRGS) in predicting the prognosis of CRC patients. Method: In this study, we retrospectively analyzed the gene expression profiles from six CRC cohorts. A total of 1,768 CRC patients with complete prognostic information were divided into the training cohort (n = 566) and two validation cohorts (n = 624 and 578, respectively). The LASSO Cox model was applied to construct a prediction model. To further validate the clinical significance of the model, we also validated the model with Genomics of Drug Sensitivity in Cancer (GDSC) and an advanced clear cell renal cell carcinoma (ccRCC) immunotherapy data set. Results: We constructed a prognostic DRGS consisting of 11 different genes to stratify patients into high- and low-risk groups. Patients in the high-risk groups had significantly worse disease-free survival (DFS) than those in the low-risk groups in all cohorts [training cohort: hazard ratio (HR) = 2.40, p < 0.001, 95% confidence interval (CI) = 1.67-3.44; validation-1: HR = 2.20, p < 0.001, 95% CI = 1.38-3.49 and validation-2 cohort: HR = 2.12, p < 0.001, 95% CI = 1.40-3.21). By validating the model with GDSC, we could see that among the chemotherapeutic drugs such as oxaliplatin, 5-fluorouracil, and irinotecan, the IC50 of the cell line in the low-risk group was lower. By validating the model with the ccRCC immunotherapy data set, we can clearly see that the overall survival (OS) of the objective response rate (ORR) with complete response (CR) and partial response (PR) in the low-risk group was the best. Conclusions: DRGS is a favorable prediction model for patients with CRC, and our model can predict the response of cell lines to chemotherapeutic agents and potentially predict the response of patients to immunotherapy.

Keywords:  DNA repair–related genes; colorectal cancer; immunotherapy; microsatellite instability; prognostic

DOI:  https://doi.org/10.3389/fgene.2022.872238
J Cancer. 2022 ;13(7): 2126-2137

BET inhibitor JQ1 enhances anti-tumor immunity and synergizes with PD-1 blockade in CRC.

Huijin Wang, Guangyao Liu, Xinghan Jin, Shenglei Song, Songyao Chen, Peiqing Zhou, Huan Li, Jianming Liang, Bo Li, Changhua Zhang, Yulong He.

  Most colorectal cancer (CRC) patients are insensitive to immune checkpoint inhibitors (ICIs) due to the immunosuppressive tumor microenvironment (TME). Epigenetic factors such as the bromo-and extraterminal domain (BET) family proteins may be responsible for the immunosuppressive microenvironment. Previous studies have shown that inhibitors of BET family proteins have the potential to remodel the immunosuppressive TME. However, data on the role of BET inhibitors in immune microenvironment in CRC remains unclear. Here, we evaluated the immunoregulatory role of JQ1, a BET inhibitor, in CRC. Transcriptome sequencing data showed that JQ1 decreased CD274 expression and increased H2Kb expression in MC38 cells. Flow cytometry assays demonstrated that JQ1 decreased cell-surface PD-L1 expression in MC38 and HCT116 cells. Moreover, JQ1 significantly increased cell-surface expression of major histocompatibility complex class I (MHC-I) in MC38 cells and HCT116 cells. Antigen-specific cytotoxic T lymphocytes (CTLs) assay demonstrated that JQ1 enhanced the MHC-I-mediated cytotoxicity of CTLs. Mouse colon cancer cell line MC38 was used to establish the syngeneic mouse tumor model. Compared with the control, JQ1 significantly inhibited tumor growth and prolonged the overall survival of the mice. Besides, JQ1 did not only inhibit tumor growth by enhancing anti-tumor immunity, but also promoted the anti-tumor effect of PD-1 antibody. In addition, our data showed that JQ1 reduced infiltration of intratumoral regulatory T cells (Treg), thus remodeling the immunosuppressive TME. Taken together, these results highlight a new approach that enhances anti-PD-1 sensitivity in CRC.

Keywords:  Colorectal cancer; JQ1; immunosuppression.; immunotherapy

DOI:  https://doi.org/10.7150/jca.69375
Cancer Discov. 2022 May 06. OF1

Cancer Cells Induce Delayed DNA Breaks to Resist Radiation Therapy.

Cancer cells resist irradiation by inducing regulated DNA breaks to prevent premature mitotic entry.

DOI: https://doi.org/10.1158/2159-8290.CD-RW2022-081
Clin Transl Oncol. 2022 May 02.

Cell-free circulating tumor DNA in colorectal cancer: a proof of concept with simplified methodology.

Javier Bosque, Carlos Guirao, Asia Ferrández, Noelia Suarez, Maria Isabel Castillejo, Diana Anguita, María Pamies, Alejandro Moya, José Luis Soto, Javier Gallego Plazas.

   BACKGROUND: Cell-free DNA analysis (cfDNA) holds promise for residual disease or tumor burden quantification in colorectal cancer, with reduced costs and diagnostic equipment compared to gold standard-specific tumor DNA (ctDNA) analysis.
METHODS: This prospective case-control study included 46 colorectal cancer patients and healthy controls to perform cfDNA quantification by fluorometry using Quantus Fluorometer (Promega, Madison, WI) and using cell-free DNA ScreenTape assay (Agilent) and 4200 TapeStation instrument (Agilent Technologies, Inc., Santa Clara, CA, USA). cfDNA quantification results were correlated with stage, clinical and histopathological features.
RESULTS: 33 localized (8 stage I, 12 stage II, and 13 stage III) and 13 advanced colorectal cancer patients were included. No differences in cfDNA quantification by fluorometry were demonstrated depending on stage or histopathological features in localized disease patients. Differences in cfDNA quantification by fluorometry could be demonstrated in patients with advanced disease depending on the presence of liver metastases and synchronous or metachronous metastatic disease. Differences in cfDNA quantification by fluorometry could be demonstrated between advanced colorectal cancer patients and both localized disease patients and healthy controls. Secondary cfDNA analysis by electrophoresis, although showing more specificity to measure ctDNA in cfDNA values, could not improve the capacity to detect differences between analyzed a groups beyond previously achieved with fluorometry.
CONCLUSION: This exploratory analysis of cfDNA based on fluorometry and electrophoresis methods showed promising results discriminating colorectal cancer and non-cancer patients, as well as different colorectal cancer stages and disease profiles. Further studies are needed to increase our knowledge and to help to overcome barriers to broader implementation and applications.

Keywords:  Cell-free; Circulating DNA; Colorectal cancer; Electrophoresis; Fluorometry

DOI:  https://doi.org/10.1007/s12094-022-02841-8
Anticancer Res. 2022 May;42(5): 2363-2374

Effectiveness of Hyperthermia as Monotherapy and Adjuvant Therapy Approaches Against an In Vitro Model of Colorectal Carcinoma.

Georgios Petrakis, Theodora Mantso, Michail I Koukourakis, Mihalis I Panayiotidis, Sotiris Botaitis.

   BACKGROUND/AIM: Despite improvement in current therapies, the 5-year overall survival rate of colorectal carcinoma is still low especially in its metastatic form. On the other hand, hyperthermia has been utilized as a cancer treatment approach to improve overall therapeutic efficacy. In the present study, we have aimed to develop an optimized hyperthermic protocol against an in vitro model of human colon carcinoma, as a single and/or adjuvant treatment approach.
MATERIALS AND METHODS: We have utilized an in vitro model of human colorectal carcinoma consisting of colorectal carcinoma (HT29, CaCo2) and normal colon epithelial (CCD841CoN) cell lines. Cells were exposed to 45°C, over 120 min, in the presence or absence of chemotherapeutic (5-Fluorouracil, Capecitabine) and targeted (Bevacizumab, Cetuximab) drugs. Cell viability levels were determined by the Alamar-blue assay while determination of cell death, reactive oxygen species (ROS) production, mitochondrial membrane depolarization (ΔΨμ) levels and cell cycle progression were performed by flow cytometry.
RESULTS: CaCo2 and HT29 cells showed a differential response towards i) cell viability, ii) cell death, iii) ROS and ΔΨμ levels as well as iv) cell cycle distribution, in the presence of hyperthermia alone (monotherapy) or in combination with the above-mentioned drugs (adjuvant therapy). Finally, normal colon epithelial (CCD841CoN) cells remained minimally affected.
CONCLUSION: We have developed an optimized experimental hyperthermic protocol, as a promising monotherapy and/or adjuvant therapy approach, with the capacity to potentiate chemotherapeutic as well as targeted drug-induced cytotoxicity against a model of colorectal carcinoma, to a variable degree.

Keywords:  5-fluorouracil; Hyperthermia; bevacizumab; cancer therapeutics; capecitabine; cetuximab; colorectal carcinoma

DOI:  https://doi.org/10.21873/anticanres.15715
Methods Mol Biol. 2022 ;2429 555-565

Orthotopic Xenografts of Colorectal Cancer Stem Cells.

Maria Laura De Angelis, Federica Francescangeli, Ann Zeuner, Marta Baiocchi.

  Cancer stem cells (CSCs) are responsible for the initiation of primary tumors and for metastasis seeding at distant organs. Therefore, they represent crucial targets for the study and preclinical testing of new antimetastatic approaches. We recently generated a molecularly characterized biobank of colorectal CSCs, isolated from individual patients and cultured in serum-free medium as multicellular spheroids. Here, we describe in detail the generation of a metastatic model of colorectal cancer based on the orthotopic injection of CSCs into the cecum serosa of immunodeficient mice. Such a model represents an excellent experimental system to investigate the cellular and molecular mechanisms involved in colorectal cancer metastasis, to analyze rare premetastatic elements such as circulating and disseminated tumor cells, and for the preclinical testing of new agents with potential antimetastatic activity.

Keywords:  Cancer stem cell-based xenografts; Cancer stem cells; In vivo preclinical models; Metastatic models; NSG mice; Orthotopic xenografts

DOI:  https://doi.org/10.1007/978-1-0716-1979-7_39
Target Oncol. 2022 May 07.

Genomic Landscape of Primary Tumor Site and Clinical Outcome for Patients with Metastatic Colorectal Cancer Receiving Standard-of-Care Chemotherapy.

Takuro Mizukami, Masaki Takahashi, Yu Sunakawa, Satoshi Yuki, Yoshinori Kagawa, Atsuo Takashima, Kyoko Kato, Hiroki Hara, Tadamichi Denda, Yoshiyuki Yamamoto, Manabu Shiozawa, Eiji Oki, Wataru Okamoto, Takayuki Yoshino, Takako Eguchi Nakajima.

BACKGROUND: Primary tumor site and genomic status are utilized for regimen selection in metastatic colorectal cancer; however, the impact on clinical practice is not well known.
OBJECTIVE: We aimed to clarify the impact of primary tumor site and genomic status on clinical practice in metastatic colorectal cancer.
METHODS: The relationship between primary tumor site, genomic alterations, and clinical outcomes was evaluated in patients with untreated metastatic colorectal cancer using real-world data of a prospective observational study, SCRUM-Japan GI-SCREEN with clinical and genomic data set in 1011 patients enrolled from February 2015 to March 2017.
RESULTS: Five hundred and sixty-one patients were eligible for this study. Patients with right-sided tumors had a significantly worse survival, left-sided tumors with wild-type RAS had favorable outcomes when treated with anti-epidermal growth factor receptor monoclonal antibodies, and cecum tumors had poor prognosis when treated with bevacizumab. The rate of gene alterations varied considerably depending on the primary site. In addition, gene alterations of KRAS, BRAF, SMAD4, or TP53 had individually different contributions to survival from site to site. KRAS, BRAF, PTEN, or SMAD4 mutations were associated with efficacy of bevacizumab or anti-epidermal growth factor receptor monoclonal antibodies.
CONCLUSIONS: Primary tumor site is a clinically useful biomarker to predict survival in patients with metastatic colorectal cancer treated with first-line chemotherapy. Moreover, the prognostic or predictive value of several gene alterations by primary tumor site should be considered in clinical practice.

DOI: https://doi.org/10.1007/s11523-022-00880-3
Exp Ther Med. 2022 Jun;23(6): 394

Oxaliplatin induces ferroptosis and oxidative stress in HT29 colorectal cancer cells by inhibiting the Nrf2 signaling pathway.

Ben Liu, Hui Wang.

  Oxaliplatin is a third-generation platinum drug that is used as first-line chemotherapy for colorectal cancer (CRC). Ferroptosis has been demonstrated to induce cell death and oxidative stress in CRC. The aim of the present study was to investigate whether oxaliplatin could exert anticancer effects on CRC by promoting ferroptosis and oxidative stress. Cell viability and apoptosis were assessed by performing Cell Counting Kit-8 and TUNEL assays, respectively, in the presence or absence of the ferroptosis inducer, erastin. Western blotting was performed to detect the levels of certain nuclear factor erythroid 2-related factor 2 (Nrf2)-associated proteins in HT29 cells treated with oxaliplatin. Furthermore, after treating cells with the Nrf2 activator, NK-252, Fe2+ was detected in cells using a commercial kit. Ferroptosis-associated protein expression was also evaluated via western blotting. Additionally, ELISA was adopted to measure the levels of oxidative stress-related factors. Following the addition of erastin, iron ion content, ferroptosis-related protein expression and the levels of oxidative stress-related factors were assayed as described previously. The results of the present study demonstrated that oxaliplatin inhibited viability and the Nrf2 signaling pathway in CRC cells. In addition, oxaliplatin promoted ferroptosis and oxidative stress in CRC cells by inhibiting the Nrf2 signaling pathway. Treatment with oxaliplatin enhanced the effects of erastin on CRC cells by promoting ferroptosis and oxidative stress and inhibiting cell viability. In conclusion, oxaliplatin induced ferroptosis and oxidative stress in CRC cells by inhibiting the Nrf2 signaling pathway.

Keywords:  colorectal cancer; ferroptosis; nuclear factor erythroid 2-related factor 2 signaling pathway; oxaliplatin; oxidative stress

DOI:  https://doi.org/10.3892/etm.2022.11321
Curr Opin Genet Dev. 2022 Apr 29. pii: S0959-437X(22)00023-5. [Epub ahead of print]74 101914

The origins of cancer cell dormancy.

Jorge Morales-Valencia, Gregory David.

Cancer cell dormancy has emerged as an important nongenetic driver of drug resistance. Dormant cells are characterized by a reversible cell cycle exit. They represent a reservoir for eventual cancer relapse, and upon reactivation, can fuel metastatic disease. Although dormant cells were originally believed to emerge from a drug-resistant pre-existing cancer subpopulation, this notion has been recently challenged. Here, we review recent evidence indicating that dormancy represents an adaptive strategy employed by cancer cells to avoid the cytotoxic effects of antitumor therapy. Furthermore, we outline the molecular pathways engaged by cancer cells to enter dormancy upon drug exposure, with a focus on cellular senescence as a driver of dormancy.

DOI: https://doi.org/10.1016/j.gde.2022.101914