Mutant Colon Cancer Cells Research Articles

Transglutaminase 2 serves as a pathogenic hub gene of KRAS mutant colon cancer based on integrated analysis.

Colon cancer is acknowledged as one of the most common malignancies worldwide, ranking third in United States regarding incidence and mortality. Notably, approximately 40% of colon cancer cases harbor oncogenic KRAS mutations, resulting in the continuous activation of epidermal growth factor receptor signaling. To investigate the key pathogenic genes in KRAS mutant colon cancer holds considerable importance. Weighted gene co-expression network analysis, in combination with additional bioinformatics analysis, were conducted to screen the key factors driving the progression of KRAS mutant colon cancer. Meanwhile, various in vitro experiments were also conducted to explore the biological function of transglutaminase 2 (TGM2). Integrated analysis demonstrated that TGM2 acted as an independent prognostic factor for progression-free survival. Immunohistochemical analysis on tissue microarrays revealed that TGM2 was associated with an elevated probability of perineural invasion in patients with KRAS mutant colon cancer. Additionally, biological roles of the key gene TGM2 was also assessed, suggesting that the downregulation of TGM2 attenuated the proliferation, invasion, and migration of the KRAS mutant colon cancer cell line. This study underscores the potential significance of TGM2 in the progression of KRAS mutant colon cancer. This insight not only offers a theoretical foundation for therapeutic approaches but also highlights the need for additional clinical trials and fundamental research to support our preliminary findings.

Tumor sensitization to PARP inhibitors by DNA damaging synthetically lethal acylfulvenes.

e15123 Background: 20-30% of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor some level of deficiency in DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DNA damaging agents. The concept of synthetic lethality has been leveraged to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful in Homologous Recombination Deficiency (HRD) cancers. LP-100 and LP-184 are acylfulvene (AF) class small molecule prodrugs that are metabolized to active compounds by prostaglandin reductase 1 (PTGR1) that is often elevated in solid tumors. AF-induced DNA lesions are likely selectively processed by repair pathways including BRCA1/2 mediated Homologous Recombination (HR). Methods: Cell Titer Glo based 10 day cell viability assays were used to generate drug IC50 values in vitro. Bliss synergy scores were computed with scores ≥ 10 reflecting synergy. Drug-induced DNA double strand breaks (DSB) were detected and quantified with STRIDE (SensiTive Recognition of Individual DNA Ends) assay in cancer cells. A subcutaneous tumor xenograft model was used to determine tumor volumes in control and drug treated mice. Results: In a BRCA2 mutant ovarian cancer cell line OVCAR3, LP-184 and olaparib co-treatment in vitro showed synergistic effects (Bliss score 10). Similarly in a BRCA2 mutant prostate cancer cell line 22Rv1, LP-100 combination with rucaparib was synergistic (Bliss score 18). 400 nM LP-184 showed a time dependent increase in DNA DSB signals in vitro, 1.7X more so in BRCA2 mutant vs wild type (WT) DLD-1 isogenic colon cancer cell lines up to 24 hours. To validate the hypothesis that AF-induced DSBs remain unresolved if exposed to and further synergize with PARPi in vivo, we tested LP-100 plus olaparib in a DU145 (BRCA2 mutant) prostate cancer xenograft model. LP-100 + olaparib treatment resulted in sustained, durable tumor regression indicating in vivo synergy whereas either agent alone did not produce tumor shrinkage. Day 42 tumor growth inhibition was 66% for 2.5 mg/kg LP-100 alone, 34% for 40 mg/kg olaparib alone and 108% for the combination. Moreover, LP-184 treatment resulted in early onset and complete tumor regression in 7 PDX models of PARPi resistant triple negative breast cancers (TNBC) harboring BRCA1/2 loss whereas olaparib/ niraparib remained entirely ineffective in these. Conclusions: AF response is influenced by tumor DNA repair competence. LP-100 and LP-184 are synthetically lethal resulting from their abilities to cause unresolvable DNA damage where tumor cells express high PTGR1 and are deficient in the HR pathway. LP-184 single agent was superior to PARPi in TNBC and LP-100 combination made an olaparib resistant tumor highly sensitive, thereby facilitating a path to extend the opportunities for both AFs and PARPis and enhance the clinical utilization of PARPi.

Abstract 412: Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer

Abstract Background: Lung cancer is one of the most common tumors, accounting for approximately 1.8 million deaths worldwide in 2020. KRAS is a frequently mutated oncogene, with mutations reported in roughly 20-25% of non-small cell lung cancer cases. Specifically, KRAS mutations at amino acid 12, resulting in a glycine to cysteine (G12C) substitution, occur in approximately 13% and 3% of lung and colon cancers, respectively, and less frequently in other solid tumors. Recently, the development of covalent KRASG12C inhibitors has shown meaningful anticancer activity in patients. However, many patients with KRASG12C do not respond and/or develop resistance to single-agent treatment. Here we investigate combination therapies that may overcome resistance and broaden patient response to KRASG12C inhibitors. Methods: In vitro preclinical KRASG12C models including lung and colon cancer cell lines were treated with a panel of small molecule and covalent KRASG12C inhibitors. The activity of single-agent versus combination treatments was measured in cell viability assays, and screen hits were validated by in vitro mechanism of action studies. Results: A curated set of 152 compounds was used in combination with screen monitoring cell-viability assays across 12 KRAS mutant lung and colon cancer cell lines. Compounds tested included kinase inhibitors targeting oncogenic signaling pathways, epigenetics modifiers, regulators of apoptosis, chemotherapeutics, and other anticancer agents. The screen identified synergy between covalent KRASG12C and several other inhibitors including those for FGFR, EGFR, and SHP2. Notably, pemigatinib, a potent and selective FGFR1-3 inhibitor, had a significantly high Bliss synergy score. Selectivity and siRNA knockdown experiments were performed to identify the specific FGFR isoform involved in synergistic anticancer activity. Inhibition of FGFR1 activity was shown to be essential, whereas an FGFR2-3 specific inhibitor demonstrated only modest activity in combination with KRASG12C inhibitors. Additionally, FGFR1 knockdown combined with KRASG12C inhibition resulted in increased drug sensitivity; in contrast, knockdown of other FGFR family members did not demonstrate similar increase in sensitivity to KRASG12C inhibition. Conclusions: Cell-viability combination screening identified strong synergy between KRASG12C covalent inhibitors and the FGFR1-3 inhibitor pemigatinib in KRASG12C cancer cell lines. The synergistic effect of pemigatinib and KRASG12C inhibition was notably strongest in mesenchymal cancer cell lines. This effect was further validated in in vitro selectivity and siRNA studies. Together, our results support pemigatinib as a promising agent for combination therapy with KRAS inhibitors in lung cancer. Citation Format: Angela Abdollahi, Margaret Favata, Darlise DiMatteo, Sean Schuette, Erika Boarder, Mark Rupar, Ricardo Macarron, Aidan Gilmartin, Hui Wang, Alejandro Amador-Arjona. Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 412.

Abstract 2698: Improved calcium phosphate delivery system cNaD to aim clinical application

Abstract Super carbonate apatite (sCA) nanoparticle is a superb calcium phosphate delivery system for miRNA and siRNA to tumors (PlosOne 2015). We reported that sCA delivered efficiently siRNA for survivin and PTBP as well as miR-340 (Mol Cancer Ther 2015, 2015) to colon tumors implanted into nude mice. MiR-4689 and miR-29b-1-5p suppressed KRAS mutated colon cancer cells (Mol Ther NA2015, 2018) and miR-302 and miR-369 realized in vivo cancer reprograming (PlosOne 2015). In every case the RNA function was fully realized. Moreover, effect of influenza vaccination was augmented by sCA plus CpG nucleic acid (NA) (Frontiers in Immunol 2018). sCA coupled with miR-29a or miR-29b prevented the DSS (dextran sodium sulfate)-induced inflammatory bowel disease (Mol Ther NA2018). We also applied the sCA-ICG complex to minimally invasive treatment of photodynamic therapy (Mol Cancer Ther 2018). Following these outstanding findings, we tried and succeeded to establish a clinically available novel calcium phosphate delivery system, named cNaD, which controls the nano-particle size and avoids accumulation in normal organs including liver and spleen. It is characterized by its ability to efficiently deliver siRNA and microRNA to cancers and provided prominent therapeutic effect in various mouse models. Safety test has been accomplished in rats and monkeys based on GLP standards. Also, the scale-up manufacturing system >1000 folds has been attained. Our cNaD system will accelerate the development of NA medicine in the world. Citation Format: Hirofumi Yamamoto. Improved calcium phosphate delivery system cNaD to aim clinical application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2698.

Oxaliplatin-loaded nanoemulsion containing Teucrium polium L. essential oil induces apoptosis in Colon cancer cell lines through ROS-mediated pathway

Oxaliplatin (Oxa)-associated adverse side effects have considerably limited the clinical use of the drug in colon cancer therapy. Mutant p53 has diverse mutational profiles in colon cancer, and it influences the potencies of various chemotherapeutic drugs, including Oxa. Thus, it would be highly beneficial to identify an alternative therapeutic strategy that not only reduces the toxicity of Oxa, but also exerts a synergistic effect against colon cancers, regardless of their p53 profiles. The present study was aimed at preparing and optimizing Teucrium polium L. essential oil nanoemulsion (TPO-NANO) and investigating its effect on the sensitivity of colon cancer cells with differences in p53 status (HCT116 wild-type and HT-29 mutant-type) to Oxa. The viability of treated cells was determined and the combination index (CI) was calculated. Morphological changes were determined under inverted microscopy, while percentage apoptosis was assayed using flow cytometry. Intracellular ROS and the protein levels of p53 and Bax were measured. The colony-forming potential of treated cells was determined using colony assay. The size of TPO-NANO was markedly increased from 12.90 ± 0.04 nm to 14.47 ± 0.53 nm after loading Oxa (p ≤ 0.05). The combination (Oxa + TPO-NANO) produced a synergetic effect in HCT116 and HT-29, with CI of 0.94 and 0.88, respectively. Microscopic examination and flow cytometric analysis revealed that cells treated with Oxa + TPO-NANO had a higher percentage of apoptosis than cells exposed to monotherapy. Cumulatively, Oxa exerted an apoptotic effect on wild or mutant p53 colon cancer cells when combined with TPO-NANO, through a mechanism involving ROS-mediated mitochondrial apoptosis.

Abstract 1626: The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells

Abstract Colorectal cancer is one of the most common cancer in the world. The mortality rate is the third among the top ten cancers. Among colorectal cancer patients, about 65-75% of patients have overexpression of epithelial growth factor receptors (EGFR). The antibody drug cetuximab that inhibits EGFR has also been developed. Moreover, overexpression of src protein is found in about 80% of patients with colorectal cancer. Higher levels of Src expression is related to the metastasis of colon cancer. Dasatinib is an oral taken tyrosine kinase inhibitor to inhibit the src phosphorylation. Therefore, in this study, the mechanism of cetuximab and dasatinib combined treatment in the orthotopic mouse model is investigated. The in vivo mechanism of EGFR and E-cadherin crosstalk in the mouse model is discussed. In this study, HT-29 colon cancer cells with BRAF V600E mutation were orthotopically injected into the cecum wall of BALB/c nude male mice. Two weeks later, cetuximab, dasatinib, and combined treatment of cetuximab and dasatinib were applied for three weeks. The tumor sizes were then recorded after sacrificed. Tumor Tissue were processed for tissue section with H&E staining. Immunohistochemistry (IHC) was applied to detect EGFR, p-Src, E-cadherin with specific antibodies. The protein expression levels were achieved by Western blot. In the results, the tumor size was smaller in cetuximab and dasatinib combined treated group. In IHC staining, the E-cadherin expression were not apparent in primary tumor site, however, were abundant in migrated cells. P-src expression was reduced in the dasatinib treated group and combined treatment group. In western blot result, the E-cadherin was increased after the treatment of dasatinib, and the p-src expression was inhibited after dasatinib treatment and combined treatment. Our results demonstrated cetuximab combined with dasatinib has the better result of suppression of BRAF mutated colon cancer cell growth and metastasis in the orthotopic mouse model. Citation Format: Yu-Cheng Weng, Yu-Lin Li, Xiang-Ling Hou, Hui Min Koo, Yi-Wen Lu, Viriya Adhiguna Winarso, Chia-Wen Lin, Wei-Ting Chao. The orthotopic mouse study of cetuximab and dasatinib treatment on BRAF mutated colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1626.

High-throughput virtual screening and preclinical analysis identifies CB-1, a novel potent dual B-Raf/c-Raf inhibitor, effective against wild and mutant variants of B-Raf expression in colorectal carcinoma.

Paradoxical Raf activation via Raf dimerization is a major drawback of wild/mutant B-Raf inhibitors. Herein, we report that CB-1 a novel, potent B-Raf/c-Raf dual inhibitor, effective against colon cancer cells, irrespective of their genetic status. High-throughput virtual screening of the ChemBridge library against wild B-Raf (B-RafWT), mutant B-Raf (B-RafV600E), and c-Raf was performed using an automated protocol with the AutoDock-VINA. Caco-2 and HT-29 cells were used. Of the 23,365 compounds screened computationally, CB-1 showed the highest binding energy towards B-RafWT with a ΔGbinding score of -13.0kcal/mol. The compound was also predicted to be effective against B-RafV600E and c-Raf molecules with ΔGbinding energies of -10.6 and -10.1kcal/mol, respectively. The compound inhibited B-RafWT, B-RafV600E and c-Raf kinases with IC50 values of 27.13, 51.70, and 40.23 nM, respectively. The GI50 value of CB-1 was 247.9 nM in B-RafWT-expressing Caco-2 cells and 352.4 nM in B-RafV600E-expressing HT-29 cells. Dose-dependent increases in total apoptosis and G1 cell cycle phase arrest was observed in CB-1-treated colon cancer cells. The compound decreased B-Raf expression in both wild and mutant colon cancer cells. CB-1, a novel, potent dual B-Raf/c-Raf inhibitor was effective against colon cancer cells bearing wild-type and mutant variants of B-Raf expression.

Digitoxin promotes apoptosis and inhibits proliferation and migration by reducing HIF-1α and STAT3 in KRAS mutant human colon cancer cells

Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor. New treatment options are needed to improve survival in patients with KRAS mutated colorectal cancer. Digitoxin is a cardiotonic drug, which has been demonstrated to exhibit anticancer effects in a number of cancers. However, the anticancer mechanisms of digitoxin in KRAS mutant human colon cancer cells remain elusive. Our result demonstrated that digitoxin but not cetuximab markedly decreased the expression of hypoxia-inducible factor-1α (HIF-1α), signal transducer and activator of transcription 3 (STAT3) and p-STAT3 protein in KRAS mutant colon cancer cells. Further analysis revealed that digitoxin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. The phosphorylation levels of ribosomal protein S6 kinase (p70S6K) and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by digitoxin. Digitoxin inhibited the expression and activation of STAT3 through upregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN), SHP1 and protein inhibitors of activated STAT3 (PIAS3) and direct binding to STAT3. Meanwhile, digitoxin inhibited HIF-1α in STAT3-independent manner in KRAS mutant colon cancer cells. Moreover, digitoxin promoted apoptosis and inhibited proliferation and migration, which was potentially mediated by suppression of HIF-1α and STAT3. We also found that digitoxin administration inhibited tumor growth in a mouse xenograft model. Taken together, our findings highlight the therapeutic potential of digitoxin for the treatment of cetuximab-resistant human colon cancer.

The Sphingosine Kinase 2 Inhibitor ABC294640 Restores the Sensitivity of BRAFV600E Mutant Colon Cancer Cells to Vemurafenib by Reducing AKT-Mediated Expression of Nucleophosmin and Translationally-Controlled Tumour Protein.

Vemurafenib (PLX4032), small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harboring BRAFV600E mutation. Unfortunately, the effect of PLX4032 in the treatment of metastatic BRAF mutated colorectal cancer (CRC) is less potent due to high incidence of fast-developing chemoresistance. It has been demonstrated that sphingolipids are important mediators of chemoresistance to various therapies in colon cancer. In this study, we will explore the role of major regulators of sphingolipid metabolism and signaling in the development of resistance to vemurafenib in BRAF mutant colon cancer cells. The obtained data revealed significantly increased expression levels of activated sphingosine kinases (SphK1 and SphK2) in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by increased expression levels of the enzymes regulating the ceramide salvage pathway, namely ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib. Pharmacological inhibition of SphK1/SphK2 activities or modulation of ceramide metabolism by exogenous C6-ceramide enhanced the anti-proliferative effect of PLX4032 in resistant RKO cells in a synergistic manner. It is important to note that the inhibition of SphK2 by ABC294640 proved effective at restoring the sensitivity of resistant cells to vemurafenib at the largest number of combinations of sub-toxic drug concentrations with minimal cytotoxicity. Furthermore, the obtained findings revealed that enhanced anti-proliferative, anti-migratory, anti-clonogenic and pro-apoptotic effects of a combination treatment with ABC294640 and PLX4032 relative to either drug alone were accompanied by the inhibition of S1P-regulated AKT activity and concomitant abrogation of AKT-mediated cellular levels of nucleophosmin and translationally-controlled tumour protein. Collectively, our study suggests the possibility of using the combination of ABC294640 and PLX4032 as a novel therapeutic approach to combat vemurafenib resistance in BRAF mutant colon cancer, which warrants additional preclinical validation studies.

Proteomic Profiling of BRAFV600E Mutant Colon Cancer Cells Reveals the Involvement of Nucleophosmin/c-Myc Axis in Modulating the Response and Resistance to BRAF Inhibition by Vemurafenib.

BRAFV600E mutations are found in approximately 10% of colorectal cancer patients and are associated with worse prognosis and poor outcomes with systemic therapies. The aim of this study was to identify novel druggable features of BRAFV600E-mutated colon cancer (CC) cells associated with the response and resistance to BRAFV600E inhibitor vemurafenib. Towards this aim, we carried out global proteomic profiling of BRAFV600E mutant vs. KRAS mutant/BRAF wild-type and double wild-type KRAS/BRAF CC cells followed by bioinformatics analyses. Validation of selected proteomic features was performed by immunohistochemistry and in silico using the TCGA database. We reveal an increased abundance and activity of nucleophosmin (NPM1) in BRAFV600E-mutated CC in vitro, in silico and in tumor tissues from colon adenocarcinoma patients and demonstrate the roles of NPM1 and its interaction partner c-Myc in conveying the resistance to vemurafenib. Pharmacological inhibition of NPM1 effectively restored the sensitivity of vemurafenib-resistant BRAF-mutated CC cells by down-regulating c-Myc expression and activity and consequently suppressing its transcriptional targets RanBP1 and phosphoserine phosphatase that regulate centrosome duplication and serine biosynthesis, respectively. Altogether, findings from this study suggest that the NPM1/c-Myc axis could represent a promising therapeutic target to thwart resistance to vemurafenib in BRAF-mutated CC.

Hsa-miR-125b Therapeutic Role in Colon Cancer Is Dependent on the Mutation Status of the TP53 Gene.

Colon cancer is the third most common cancer type worldwide and is highly dependent on DNA mutations that progressively appear and accumulate in the normal colon epithelium. Mutations in the TP53 gene appear in approximately half of these patients and have significant implications in disease progression and response to therapy. miR-125b-5p is a controversial microRNA with a dual role in cancer that has been reported to target specifically TP53 in colon adenocarcinomas. Our study investigated the differential therapeutic effect of miR-125b-5p replacement in colon cancer based on the TP53 mutation status of colon cancer cell lines. In TP53 mutated models, miR-125b-5p overexpression slows cancer cells’ malignant behavior by inhibiting the invasion/migration and colony formation capacity via direct downregulation of mutated TP53. In TP53 wild type cells, the exogenous modulation of miR-125b-5p did not significantly affect the molecular and phenotypic profile. In conclusion, our data show that miR-125b-5p has an anti-cancer effect only in TP53 mutated colon cancer cells, explaining partially the dual behavior of this microRNA in malignant pathologies.

Equipping Natural Killer Cells with Cetuximab through Metabolic Glycoengineering and Bioorthogonal Reaction for Targeted Treatment of KRAS Mutant Colorectal Cancer.

While Cetuximab can be used to treat KRAS wild-type colon cancer cells by targeting EGFR and inhibiting the activation of downstream signaling pathways, it exhibits little therapeutic effect on KRAS mutant colon cancer cells. Natural killer (NK) cells are a class of powerful immune cells with anticancer activities. However, NK cells typically lack inherent tumor targeting abilities. Here, a new method is established to bestow NK-92 cells with tumor targeting abilities by installing cetuximab on the cell surface. Through metabolic glycoengineering, azide groups were introduced onto the surface of NK-92 cells. Bioorthogonal strain promoted the azide-alkyne cycloaddition click reaction of engineered NK-92 cells with alkyne modified cetuximab functionalized NK cells with the antibody. The resulting NK-92 cells were significantly more effective than the parent NK-92 cells in protecting against tumor development in a KRAS mutant mouse tumor model resistant to cetuximab treatment. Thus, NK cell functionalization with antibodies enabled by metabolic glycoengineering is a promising strategy to enhance anticancer immune therapy.

TRIM29 Reverses Oxaliplatin Resistance of P53 Mutant Colon Cancer Cell.

Background Oxaliplatin is the first-choice chemotherapy method for patients with advanced colon cancer. However, its resistance leads to treatment failure for many patients. In our experiments, we aim to elucidate the associations among TRIM29 protein, mutant P53, and the resistance of colon cancer cells to oxaliplatin. Methods HCT116 and HT-29 cells were cultured and transfected with plasmids pIRES2-ZsGreen1-TRIM29-flag. Western blot and real-time qRT-PCR were utilized to examine the protein and mRNA expressions of TRIM29 and other related markers, respectively. MTT assay was utilized to determine the cell growth rate and generate the inhibition curve. Continuous culture in low-concentration oxaliplatin was conducted to construct oxaliplatin-resistant cell lines. The coimmunoprecipitation method and immunofluorescence detection were used to examine the interaction between TRIM29 and mutant P53 protein in HT29 cells. Results We successfully transfected pIRES2-ZsGreen1-TRIM29-flag into HCT116 and HT29 cells, which were utilized in the whole experiments. TRIM29 significantly increased the sensitivity of P53 mutant colon cancer cell HT29 to oxaliplatin. The oxaliplatin-resistant model of P53 mutant colon cancer cell HT29 was successfully constructed. TRIM29 physically bound with mutant P53 and retained it in the cytoplasm from the nucleus, which inhibited its transcription function of downstream genes such as MDR1. In addition, TRIM29 successfully reversed the resistance of HT29-OX resistant cell model to oxaliplatin. Conclusion In mutant P53 colon cancer cell HT29, TRIM29 greatly increased the sensitivity of HT29 to oxaliplatin and reverse oxaliplatin resistance. The underlying mechanism is TRIM29 may increase the sensitivity of HT29 to oxaliplatin by blocking the transcriptional function of mutant P53, which inhibits the transcription function of its downstream gene such as MDR1.

Autophagy Flux Correlates with Upregulation of AKT-1 in RAS Mutated Colon Cancer Cells

Background: The AKT/PKB (protein kinase B) kinase is the main regulator of autophagy in mammalian cells, which consists of three isoforms, including AKT-1, AKT-2, and AKT-3. Rat sarcoma viral oncogene homolog (RAS), known as the most frequently mutated oncogene in colorectal cancers, is one of the major activators of AKT signaling. However, the relationship between AKT isoforms expression and autophagy level in RAS-driven cancer cells has not been fully investigated. Method: In this experimental in vitro study, RAS mutated colon cancer cell lines (HCT116, SW480, and LS180) and HT29 cells, which are the wild type of RAS, were cultured and real-time polymerase chain reaction (RT-PCR) was utilized to determine the mRNA level of AKT-1, AKT-2, and autophagy markers, including microtubule-associated protein 1 light chain-3B (LC3B) and p62/sequestosome-1 (p62). In addition, Western blotting was performed to assess the protein expression of p62 and LC3B lipidation. Results: We found that RAS mutated colon cancer cells up-regulate basal autophagy. Moreover, highly expressed AKT-1 was observed in RAS mutated colon cancer cells. However, no significant differences were found in AKT-2 expression between RAS-driven cells and HT29 cells. Conclusion: Our obtained data suggested that RAS-driven colon cancer cells regulated the autophagy machinery, possibly, through the upregulation of AKT-1 isoform.

Retraction Note: JNK confers 5-fluorouracil resistance in p53-deficient and mutant p53-expressing colon cancer cells by inducing survival autophagy

Editor's Note: this Article has been retracted; the Retraction Note is available at https://doi.org/10.1038/s41598-021-82870-5.

Selective killing of cancer cells harboring mutant RAS by concomitant inhibition of NADPH oxidase and glutathione biosynthesis

Oncogenic RAS is a critical driver for the initiation and progression of several types of cancers. However, effective therapeutic strategies by targeting RAS, in particular RASG12D and RASG12V, and associated downstream pathways have been so far unsuccessful. Treatment of oncogenic RAS-ravaged cancer patients remains a currently unmet clinical need. Consistent with a major role in cancer metabolism, oncogenic RAS activation elevates both reactive oxygen species (ROS)-generating NADPH oxidase (NOX) activity and ROS-scavenging glutathione biosynthesis. At a certain threshold, the heightened oxidative stress and antioxidant capability achieve a higher level of redox balance, on which cancer cells depend to gain a selective advantage on survival and proliferation. However, this prominent metabolic feature may irrevocably render cancer cells vulnerable to concurrent inhibition of both NOX activity and glutathione biosynthesis, which may be exploited as a novel therapeutic strategy. In this report, we test this hypothesis by treating the HRASG12V-transformed ovarian epithelial cells, mutant KRAS-harboring pancreatic and colon cancer cells of mouse and human origins, as well as cancer xenografts, with diphenyleneiodonium (DPI) and buthionine sulfoximine (BSO) combination, which inhibit NOX activity and glutathione biosynthesis, respectively. Our results demonstrate that concomitant targeting of NOX and glutathione biosynthesis induces a highly potent lethality to cancer cells harboring oncogenic RAS. Therefore, our studies provide a novel strategy against RAS-bearing cancers that warrants further mechanistic and translational investigation.

Dysregulated Phosphorylation of p53, Autophagy and Stemness Attributes the Mutant p53 Harboring Colon Cancer Cells Impaired Sensitivity to Oxaliplatin.

Colorectal cancer (CRC) forms one of the highest ranked cancer types in the world with its increasing incidence and mortality rates despite the advancement in cancer therapeutics. About 50% of human CRCs are reported to have defective p53 expression resultant of TP53 gene mutation often contributing to drug resistance. The current study was aimed to investigate the response of wild-type TP53 harboring HCT 116 and mutant TP53 harboring HT 29 colon cancer cells to chemotherapeutic drug oxaliplatin (OX) and to elucidate the underlying molecular mechanisms of sensitivity/resistance in correlation to their p53 status. OX inhibited growth of wild-type p53-harboring colon cancer cells via p53/p21-Bax mediated apoptosis. Our study revealed that dysregulated phosphorylation of p53, autophagy as well as cancer stemness attributes the mutant p53-harboring colon cancer cells impaired sensitivity to OX.

Interleukin-1 receptor antagonist enhances chemosensitivity to fluorouracil in treatment of Kras mutant colon cancer.

BACKGROUNDKras mutant colon cancer shows abnormal activation of the nuclear factor kappa-B (NF-κB) pathway, resulting in the proliferation of tumor cells. Treatment with fluorouracil (5-FU) might not achieve the expected inhibition of proliferation of malignant cells based on the fluorouracil-induced activation of the NF-κB pathway.AIMTo detect whether interleukin (IL)-1 receptor antagonist (IL-1RA) could increase the chemosensitivity to 5-FU by decreasing the activation of the NF-κB pathway and reducing the proliferation of colon cancer cells.METHODSWestern blot analysis was performed to detect the persistent activation of the NF-κB pathway in colon cancer cell lines. Reverse transcription-polymerase chain reaction was used to detect the IL-1RA-reduced expression levels of IL-6, IL-8, IL-17, IL-21 and TLR4 in colon cancer cell lines. We used a xenograft nude mouse model to demonstrate the downregulation of the NF-κB pathway by blocking the NF-κB-regulated IL-1α feedforward loop, which could increase the efficacy of chemotherapeutic agents in inhibiting tumor cell growth.RESULTSIL-1 receptor antagonist could decrease the expression of IL-1α and IL-1β and downregulate the activity of the NF-κB pathway in Kras mutant colon cancer cells. Treatment with 5-FU combined with IL-1RA could increase the chemosensitivity of the SW620 cell line, and decreased expression of the TAK1/NF-κB and MEK pathways resulted in limited proliferation in the SW620 cell line.CONCLUSIONAdjuvant chemotherapy with IL-1RA and 5-FU has a stronger effect than single chemotherapeutic drugs. IL-1RA combined with fluorouracil could be a potential neoadjuvant chemotherapy in the clinic.

Application of trastuzumab emtansine in HER-2-positive and KRAS/BRAF-mutated colon cancer cells.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) for the treatment of human epidermal growth factor receptor 2 (HER-2)-positive breast cancer. T-DM1 is based on the trastuzumab antibody and delivers a toxic agent into breast cancer cells through endocytic mechanism. This study evaluated whether T-DM1 can be used in HER-2-positive colon cancer cells which harbour KRAS/ BRAF mutation with limited treatment. LS174T and HT-29 which are KRAS and BRAF mutant HER-2-positive colon cancer cells were used in this study. Cells were first treated with T-DM1; cetuximab and trastuzumab were applied for comparison, the effect of drug sensitivity was determined. Cells were then transfected with plasmid to overexpress HER-2 or the endocytic protein, caveolin-1 or furthermore pretreated with metformin to examine the effect of T-DM1 efficacy. Finally, a xenograft mouse model was used to evaluate the drug efficacy in vivo. The results showed that T-DM1 had better inhibitory effect than cetuximab and trastuzumab on LS174T and HT-29 cells. HER-2 or caveolin-1 overexpression with plasmid in the cells to increase T-DM1 recognition or internalization can increase the sensitivity to T-DM1. When cells were pretreated with metformin, caveolin-1 expression was induced and promoted T-DM1 uptake and enhanced cell toxicity. In xenograft mouse model, combined treatment of T-DM1 and metformin had apparent inhibitory effect on subcutaneous tumour growth. The results of this study suggested that T-DM1 has potential in the treatment of HER-2-positive colon cancer cells, and application of metformin has therapeutic benefits during T-DM1 treatment.

Phase II study of lamivudine in p53 mutant metastatic colorectal cancer (mCRC).

149 Background: Non-coding repeat RNAs in cancers are pervasive and “mimic” viruses with activation of pattern recognition receptors and the innate immune response. Many repeat RNAs replicate in cancer genomes through a reverse transcriptional intermediate analogous to retroviruses. Nucleoside reverse transcriptase inhibitors (NRTIs) block this retroviral life cycle to increases repeat RNAs in p53 mutant colon cancer cell cancer lines. We initiated a Phase 2 study of lamivudine (3TC) in TP53 mutant mCRC. Methods: Two-stage design with target accrual of 20 patients (pts) in stage 1 and total of 32. Eligibility: pts with p53 mutant refractory mCRC with progression on or intolerance to 5FU, oxaliplatin and irinotecan and anti-EGFR if RAS WT. RNA sequencing was performed on pre-treatment (tx) and on tx biopsy to evaluate for repeat RNA expression and expression of other genes linked to 3TC response/resistance. Radiation was allowed. 9 pts were treated with 3TC 150 mg po bid for 28-day cycles, the maximum FDA approved dose of 3TC in HIV. Subsequent pts were treated at 600 mg po bid, previously tested in P1 trials. Tumor assessments were performed every 8 weeks until documented disease progression by RECIST 1.1 criteria or drug intolerance. Results: 29/32 pts have been treated. Median age: 60 yrs. (27-82) 18 males, 11 females. 2/ 9 (22%) pts on standard 3TC dosing had stable disease (SD) on single agent 3TC with a duration of tx of 169 and 167 days, respectively. Both pts had an initial drop in CEA upon initiation of 3TC. Of the next 20 pts on high dose 3TC, 19 were evaluable. 4 had SD, for 110, 159, 130 and 228+ days. 14 pts had tx-related adverse events (TRAE). 1 pt with a definite Grade 1 TRAE (fatigue). No pts with Grade ≥3 TRAEs. We obtained pre-tx fresh frozen biopsies on 24/29 pts. Of those with SD, 4 had biopsies and differential expression identified significantly higher HSATII repeat RNA in pts with SD compared to PD. There was an association of decreased epigenetic gene expression in HSATII repeat RNA high tumors. Conclusions: This proof-of-concept study demonstrates the safety and activity of single-agent 3TC. Repeat RNA levels appear to correlate with clinical benefit and can be measured in biopsies. Further combination studies and correlatives are planned. Clinical trial information: NCT03144804.