KRAS Expression Research Articles

Single-cell RNA sequencing reveals the mechanism of PI3K/AKT/mTOR signaling pathway activation in lung adenocarcinoma by KRAS mutation.

Aberrant activation of the phosphatidlinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway has been shown to play an important role in lung adenocarcinoma (LUAD). The effect of KRAS mutations, one of the important signatures of LUAD, on the PI3K/AKT/mTOR pathway in LUAD remains unclear. The Seurat package and principal component analysis were used for cell categorization of single-cell RNA sequencing data of LUAD. The AUCell score was used to assess the activity of the PI3K/AKT/mTOR pathway. Meanwhile, using the gene expression profiles and mutation profiles in the The Cancer Genome Atlas dataset, LUAD patients were categorized into KRAS-mutant (KRAS-MT) and KRAS-wild-types (KRAS-WT), and the corresponding enrichment scores were calculated using gene set enrichment analysis analysis. Finally, the subpopulation of cells with the highest pathway activity was identified, the copy number variation profile of this subpopulation was inscribed using the inferCNV package and the CMap database was utilized to make predictions for drugs targeting this subpopulation. There is higher PI3K/AKT/mTOR pathway activity in LUAD epithelial cells with KRAS mutations, and high expression of KRAS, PIK3CA, AKT1 and PDPK1. In particular, we found significantly higher levels of pathway activity and associated gene expression in KRAS-MT than in KRAS-WT. We identified the highest pathway activity on a subpopulation of GRB2+ epithelial cells and the presence of amplified genes within its pathway. Finally, drugs were able to target GRB2+ epithelial cell subpopulations, such as wortmannin, palbociclib and angiogenesis inhibitor. The present study provides a basic theory for the activation of the PI3K/AKT/mTOR signaling pathway as a result of KRAS mutations.

KRAS modulates immune infiltration levels and survival outcomes in patients with lung adenocarcinoma

The murine sarcoma virus oncogene (KRAS) is a key gene associated with tumorigenesis and chemotherapy resistance. However, little is known about the molecular mechanisms and immune infiltration of RASs in lung adenocarcinoma. Gene Expression Profiling Interaction Analysis was used for RASs expression analysis, and Kaplan–Meier analysis was used to analyze the potential of RASs in clinical prognosis. The effect of KRAS on immune infiltration was analyzed by TIMER. In addition, the correlation between KRAS expression and molecular mechanisms was investigated by TIMER and Cancer Single-cell State Atlas (Cancer SEA). KRAS expression levels were associated with good prognosis and tumor progression. Furthermore, KRAS expression correlates with several immune cell markers and regulates tumorigenesis. KRAS expression is involved in the regulation of multiple oncogenes and tumorigenesis, especially in the prognosis and immune infiltration of lung adenocarcinoma.

Inhalable Bottlebrush Polymer Bioconjugates as Vectors for Efficient Pulmonary Delivery of Oligonucleotides.

Antisense oligonucleotides hold therapeutic promise for various lung disorders, but their efficacy is limited by suboptimal delivery. To address this challenge, we explored the use of inhaled bottlebrush polymer-DNA conjugates, named pacDNA, as a delivery strategy. Inhaled pacDNA exhibits superior mucus penetration, achieving a uniform and sustained lung distribution in mice. Targeting the 5' splice site of an aberrant enhanced green fluorescence protein (EGFP) pre-mRNA in EGFP-654 mice, inhaled pacDNA more efficiently corrects splicing than a B-peptide conjugate and restores EGFP expression in the lung. Additionally, in an orthotopic NCI-H358 non-small-cell lung tumor mouse model, inhaled pacDNA targeting wild-type KRAS mRNA effectively suppresses KRAS expression and inhibits lung tumor growth, requiring a substantially lower dosage compared to intravenously injected pacDNA. These findings demonstrate the potential of bottlebrush polymer-DNA conjugates as a promising agent for enhanced oligonucleotide therapy in the lung and advancing the treatment landscape for lung disorders.

Circular RNA hsa_circ_0001846 facilitates the malignant behaviors of pancreatic cancer by sponging miR-204-3p and upregulating KRAS expression

Pancreatic cancer (PC) is mainly derived from the exocrine pancreatic ductal epithelial cells, and it is strongly aggressive malignant tumor. Due to its insidious onset and the lack of effective diagnostic biomarkers, PC currently remains one of the main causes of cancer-related mortality worldwide. Recent studies have found that hsa_circ_0001846 is involved in the progression of multiple cancers and has the potential to become biomarkers, but its function and mechanism in PC remains unclear. We found by qRT-PCR experiments that hsa_circ_0001846 was upregulated in PC cells and tissues, while circBase, Sanger sequencing, agarose gel electrophoresis and FISH experiments identified the splicing site, ring structure and cellular localization of hsa_circ_0001846. Various functional experiments by using the construction of small interfering RNA targeting hsa_circ_0001846 and overexpression plasmid demonstrated that hsa_circ_0001846 promoted the proliferation, migration and invasion of PC cells. Moreover, the tumor weight and volume of nude mice were significantly reduced after the stable knockdown of hsa_circ_0001846. In the mechanism exploration, RNA pull-down experiments and dual-luciferase experiments helped us to determine that hsa_circ_0001846 regulated the KRAS expression by sponging miR-204-3p in PC, thus playing a pro-cancer role. In this study, the effect of miR-204-3p on PC was also explored for the first time, and we found that knockdown of miR-204-3p reversed the tumor suppressive effect caused by silencing hsa_circ_0001846, and silencing KRAS also rescued the pro-cancer effect caused by overexpression of hsa_circ_0001846. In conclusion, our study revealed the pro-cancer role of hsa_circ_0001846 in PC, and for the first time identified the mechanism that hsa_circ_0001846 regulated KRAS by sponging miR-204-3p to promote PC progression and had the potential to become a cancer biomarker.

Abstract A092: Determinants of sensitivity to BI KRASmulti inhibitor using high-throughput in-vitro drug screens

Abstract BI 3706674 is a novel small molecule inhibitor targeting KRAS in its inactive (GDP-bound) state and is currently undergoing IND enabling studies. Here, we assess the sensitivity of BI 3706674 in a large panel of cancer cell lines with the aim to identify biomarkers predictive of patient response. We employ two complementary, high-throughput cell viability screening setups: the pooled PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) platform (868 cancer cell lines representing 80 tumor types) and a well-based custom screen performed in collaboration with Horizon Discovery (292 cell lines representing 32 tumor types). Based on correlation with publicly available data of over 1464 drug sensitivity profiles, the top 10 correlated drugs (ranked by Pearson correlation) are exclusively MEK inhibitors. This analysis shows that compounds with high similarity in their sensitivity profile to our KRAS inhibitor target proteins in the MAPK pathway. Correlation of drug sensitivity data with CRISPR gene dependency data for KRAS showed that drug selectivity of BI 3706674 is specific for KRAS (Pearson R = -0.49) compared to other members of the RAS family, such as HRAS or NRAS (R=0.03 & R=0.16). BI 3706674 shows sensitivity across a wide range of KRAS alleles (KRAS wild-type (wt) copy number amplification, G12V, G12C, G13D, G12D, G12A, Q61H) with the highest sensitivity for cell lines with KRAS wt amplifications (relative copy number of > 10) followed by cell lines with a KRAS G12V and G12C mutant alleles. Efficacy was observed in 8/9 cell lines with KRAS wt relative copy number of > 10 (sensitivity threshold of 1- AUC = 0.25) in the PRISM screen, emphasizing the utility of KRAS copy number as a predictive biomarker for drug response. Furthermore, KRAS copy number amplifications and KRAS expression are highly correlated features across cell lines (Pearson R = 0.72, P=2.2e-16), indicating that both KRAS copy number amplification and KRAS expression could serve as sensitivity biomarkers for BI 3706674. In this study, we show that BI 3706674 is a potent and selective KRAS inhibitor and that KRAS copy number alterations represent a highly predictive biomarker. Conclusively, high-throughput drug screens are powerful tools to define and further refine biomarkers and study drug mechanism of actions. Citation Format: Fiorella Schischlik, Antonio Tedeschi, Dorothea Rudolph, Daniel Gerlach, Birgit Wilding, Matthias Treu, Julian Fuchs, Lorenz Herdeis, Joachim Broeker, Tobias Wunberg, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Darryl McConnell, Mark Pearson, Norbert Kraut, Christian Haslinger, Jesse Lipp. Determinants of sensitivity to BI KRASmulti inhibitor using high-throughput in-vitro drug screens [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A092.

Abstract B093: Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent

Abstract KRAS is the most frequently mutated oncoprotein in human cancers. Although long considered “undruggable”, recent breakthroughs in medicinal chemistry have led to FDA-approval of the KRAS(G12C) mutation-specific inhibitors sotorasib and adagrasib for the treatment of KRAS(G12C)-positive non-small cell lung cancer. However, illustrating the need to develop additional novel agents targeting mutated KRAS, the 5-year relative survival rate for pancreatic cancer, which is commonly associated with a variety of different KRAS mutations, is only 12% (all SEER stages) due to poor early detection and a lack of effective treatments. In particular, KRAS(G12D) is the most common KRAS mutation, being found in 37% of pancreatic ductal adenocarcinomas (PDACs), as well as in 12.5% of colorectal cancer and 4.9% of lung adenocarcinoma (LUAD) patients. MRTX1133 is a highly-selective, non-covalent KRAS(G12D) inhibitor that has recently entered a phase 1 clinical trial. However, here we show that exposure of KRAS(G12D)-mutated PDAC (PANC-1 and AsPC-1) and LUAD (SK-LU-1) cell lines and a patient-derived organoid (PDO) PDAC model (RPAN001) to MRTX1133 resulted in varying degrees of in vitro efficacy. Decreased downstream KRAS signaling in the form of reduced phospho-ERK1/2 levels was observed to rapidly recover within 24 hours of treatment with 500 nM MRTX1133. Moreover, this rebound coincided with increased expression of KRAS, NRAS andHRAS mRNAs. Concurrently, activation of the receptor tyrosine kinases (RTKs) EGFR and MET was observed in PANC-1 and SK-LU-1 cells that displayed innate resistance to MRTX1133, while the sensitive AsPC-1 cells showed no such RTK activation. These results suggest that targeting of both KRAS(G12D) and RTKs may be needed to treat KRAS(G12D) inhibitor-resistant cancers. To address this, we employed a novel hetero-bispecific CHAMP molecule, RNK08179, that simultaneously targets both KRAS(G12D) and HSP90, an RTK-regulating chaperone protein. RNK08179 treatment demonstrated a striking reduction in phospho-ERK1/2 levels, RTK activation and cell viability in MRTX1133-resistant PANC-1 and SK-LU-1 cells. Furthermore, similar efficacy was observed in the MRTX1133-resistant RPAN001 PDO model. In summary, RNK08179 displayed promising efficacy in cancer models harboring KRAS(G12D) by suppressing both mutated KRAS and HSP90-supported RTK signaling. Citation Format: Ines Pulido, Qiyue Luan, Chenghao Yin, Zimo Yang, Jinhua Lin, Yaya Wang, Yuetong Sun, Chuche Liu, Haoxin Zhou, Marek Massad, Ian Papautsky, Thomas L. Prince, Guoqiang Wang, Kevin P. Foley, Weiwen Ying, Takeshi Shimamura. Treating KRAS(G12D) inhibitor resistance using a KRAS- and HSP90 chaperone-targeted hetero-bispecific small molecule agent [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B093.

Genotoxicity and oxidative stress induction by calcium hydroxide, calcium titanate or/and yttrium oxide nanoparticles in mice

Intensive uses of Calcium hydroxide (Ca(OH)2NPs), calcium titanate (CaTiO3NPs) and yttrium oxide (Y2O3NPs) nanoparticles increase their environmental release and human exposure separately or together through contaminated air, water and food. However, too limited data are available on their genotoxicity. Therefore, this study explored the effect of Ca(OH)2NPs, CaTiO3NPs or/and Y2O3NPs administration on the genotoxicityand oxidative stress induction in mice hepatic tissue. Mice were orally administered Ca(OH)2NPs, CaTiO3NPs and Y2O3NPs separately or simultaneously together at a dose level of 50 mg/kg b.w. for two successive weeks (3 days per week). Marked induction of DNA damage noticed after oral administration of Ca(OH)2NPs or CaTiO3NPs alone together with high Ca(OH)2NPs induced reactive oxygen species (ROS) generation and a slight CaTiO3NPs induced ROS production were highly decreased after simultaneous coadministration of administration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs up to the negative control level. Oral administration of Y2O3NPs alone also did not cause observable changes in the genomic DNA integrity and the ROS generation level compared to the negative control levels. Similarly, significant elevations in P53 gene expression and high reductions in Kras and HSP-70 genes expression were observed only after administration of Ca(OH)2NPs alone, while, remarkable increases in the Kras and HSP-70 genes expression and non-significant changes in p53 gene expression were noticed after administration of CaTiO3NPs and Y2O3NPs separately or simultaneously together with Ca(OH)2NPs. Conclusion: Ca(OH)2NPs exhibited the highest genotoxic effect through oxidative stress induction and disruption of apoptotic (p53 and Kras) and protective (HSP-70) genes expression. Slight DNA damage was noticed after CaTiO3NPs administration. However, administration of Y2O3NPs alone was non-genotoxic and coadministration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs restored genomic DNA integrity and normal expression of apoptotic p53 and protective HSP-70 genes disrupted by Ca(OH)2NPs and CaTiO3NPs. Thus co-administration of Y2O3NPs with Ca(OH)2NPs and CaTiO3NPs is recommended to counter Ca(OH)2NPs and CaTiO3NPs induced genotoxicity and oxidative stress.

Association of KRAS, NRAS, BRAF and PIK3CA gene mutations with clinicopathological features, prognosis and ring finger protein 215 expression in patients with colorectal cancer.

The relationships of KRAS, NRAS, BRAF and PIK3CA gene mutations with the clinicopathological features and prognosis of colorectal cancer (CRC) in patient are lacking. Furthermore, the role of ring finger protein 215 (RNF215) in CRC patients with KRAS, NRAS, BRAF and PIK3CA mutations remains unclear. In the present study, 182 surgical resection specimens from patients with primary CRC for retrospective analysis, were collected. KRAS/NRAS/BRAF/PIK3CA gene mutations were confirmed by an amplification-refractory mutation system. Immunohistochemistry (IHC) was conducted to confirm KRAS, NRAS, BRAF and PIK3CA protein expression. RNF215 expression in patients with CRC was evaluated using TIMER 2.0 database and IHC. The individual mutation rates of KRAS, NRAS, BRAF and PIK3CA were 40.7% (74/182), 4.4% (8/182), 4.4% (8/182) and 3.3% (6/182), respectively. The KRAS exon 2 mutation rate was the highest (61.5%, 64/104), and these mutations mainly occurred at codons 12 and 13. KRAS/NRAS/BRAF/PIK3CA wild-type CRC patients had significantly longer overall survival and disease-free survival than mutated KRAS/NRAS/BRAF/PIK3CA CRC patients (P<0.05). Overall, 45.4% (5/11) of patients with PIK3CA mutations had concomitant KRAS mutations. The KRAS/NRAS/BRAF/PIK3CA gene mutation rate in patients with lymph node metastasis (76.1%, 35/46) was significantly higher than that in patients without lymph node metastasis (50.8%, 69/136) (P=0.0027). There were no significant differences in IHC expression between patients with and without KRAS, NRAS, BRAF and PIK3CA mutations (P>0.05). The TIMER 2.0 analysis showed that RNF215 expression was significantly higher in the mutated BRAF group than in the wild-type BRAF group in CRC (P<0.05). In conclusion, KRAS is the most commonly mutated gene, and KRAS mutations may be a poor prognostic factor for patients with CRC. KRAS wild-type patient resistance may be related to PIK3CA gene mutations, although this needs further verification in larger cohorts. BRAF mutations may be associated with RNF215 expression in patients with CRC.

Defining the Functional Influence of Endothelial Cell–Expressed Oncogenic Activating Mutations on Vascular Morphogenesis and Capillary Assembly

This study sought to define key molecules and signals controlling major steps in vascular morphogenesis, and how these signals regulate pericyte recruitment and pericyte-induced basement membrane deposition. The morphogenic impact of endothelial cell (EC) expression of activating mutants of Kirsten rat sarcoma virus (kRas), mitogen-activated protein kinase 1 (Mek1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Akt serine/threonine kinase 1 (Akt1), Ras homolog enriched in brain (Rheb) Janus kinase 2 (Jak2), or signal transducer and activator of transcription 3 (Stat3) expression versus controls was evaluated, along with EC signaling events, pharmacologic inhibitor assays, and siRNA suppression experiments. Primary stimulators of EC lumen formation included kRas, Akt1, and Mek1, whereas PIK3CA and Akt1 stimulated a specialized type of cystic lumen formation. In contrast, the key drivers of EC sprouting behavior were Jak2, Stat3, Mek1, PIK3CA, and mammalian target of rapamycin (mTor). These conclusions are further supported by pharmacologic inhibitor and siRNA suppression experiments. EC expression of active Akt1, kRas, and PIK3CA led to markedly dysregulated lumen formation coupled to strongly inhibited pericyte recruitment and basement membrane deposition. For example, activated Akt1 expression in ECs excessively stimulated lumen formation, decreased EC sprouting behavior, and showed minimal pericyte recruitment with reduced mRNA expression of platelet-derived growth factor-BB, platelet-derived growth factor-DD, and endothelin-1, critical EC-derived factors known to stimulate pericyte invasion. The study identified key signals controlling fundamental steps in capillary morphogenesis and maturation and provided mechanistic details on why EC activating mutations induced a capillary deficiency state with abnormal lumens, impaired pericyte recruitment, and basement deposition: predisposing stimuli for the development of vascular malformations.

The ASH1L-AS1-ASH1L axis controls NME1-mediated activation of the RAS signaling in gastric cancer.

Gastric cancer (GC) is one of the most leading cause of malignancies. However, the molecular mechanisms underlying stomach carcinogenesis remain incompletely understood. Dysregulated genetic and epigenetic alternations significantly contribute to GC development. Here, we report that ASH1L and its antisense lncRNA ASH1L-AS1, which are transcribed from the most significant GC-risk signal at 1q22, act as novel oncogenes. The high levels of ASH1L or lncRNA ASH1L-AS1 expression in GC specimens are associated with worse prognosis of patients. In line with this, ASH1L and ASH1L-AS1 are functionally important in promoting GC disease progression. LncRNA ASH1L-AS1 up-regulates ASH1L transcription, increases histone methyltransferase ASH1L expression and elevates genome-wide H3K4me3 modification levels in GC cells. Furthermore, ASH1L-AS1 directly interacts with transcription factor NME1 protein to form the ASH1L-AS1-NME1 ribonucleoprotein, which transcriptionally promotes expression of ASH1L, ASH1L-AS1, KRAS and RAF1, and activates the RAS signaling pathway in GC cells. Taken together, our data demonstrated that the ASH1L-AS1-ASH1L regulatory axis controls histone modification reprogram and activation of the RAS signaling in cancers. Thus, ASH1L-AS1 might be a novel targets of GC therapeutics and diagnosis in the clinic.

Crocin Suppresses Colorectal Cancer Cell Proliferation by Regulating miR-143/145 and KRAS/RREB1 Pathways.

As a chemoprevention agent, crocin effectively decreases the risk of human cancers, including colorectal cancer (CRC). However, the mechanism underlying the anti-cancer effects of crocin is not entirely explained. Considering that in this study, we investigated the crocin effect on miR-143/145 and related signaling pathways in CRC cells. HCT-116 and HT-29 CRC cells were treated with different concentrations of crocin and then were subjected to MTT and qRT-PCR assays to investigate cell viability and miR-143/miR-145, KRAS, and RREB1 expression, respectively. Also, western blotting was performed to evaluate gene expression at protein levels. Our results showed that treating CRC cells with crocin decreases cell viability by upregulating miR-143/145 expression and reducing KRAS and RREB1 expression dose-dependently. These effects on gene expression in CRC cells were reversed by removing crocin from the media after 48 h. Furthermore, western blotting results exhibited that crocin significantly reduced the protein expression of KRAS and RREB1. Also, it was found that treatment of CRC cells by crocin led to the inactivation of AKT by decreasing its phosphorylation. This study suggests that crocin may inhibit CRC cell proliferation by modulating KRAS, REEB1, and AKT signaling pathways mediated through miR-143/145 upregulation.

CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells.

Although the RAS oncogene has been extensively studied, new aspects concerning its role and regulation in normal biology and cancer continue to be discovered. Recently, others and we have shown that the mechanistic Target of Rapamycin Complex 2 (mTORC2) is a Ras effector in Dictyostelium and mammalian cells. mTORC2 plays evolutionarily conserved roles in cell survival and migration and has been linked to tumorigenesis. Because RAS is often mutated in lung cancer, we investigated whether a Ras-mTORC2 pathway contributes to enhancing the migration of lung cancer cells expressing oncogenic Ras. We used A549 cells and CRISPR/Cas9 to revert the cells' KRAS G12S mutation to wild-type and establish A549 revertant (REV) cell lines, which we then used to evaluate the Ras-mediated regulation of mTORC2 and cell migration. Interestingly, our results suggest that K-Ras and mTORC2 promote A549 cell migration but as part of different pathways and independently of Ras's mutational status. Moreover, further characterization of the A549REV cells revealed that loss of mutant K-Ras expression for the wild-type protein leads to an increase in cell growth and proliferation, suggesting that the A549 cells have low KRAS-mutant dependency and that recovering expression of wild-type K-Ras protein increases these cells tumorigenic potential.

Let-7g Upregulation Attenuated the KRAS-PI3K-Rac1-Akt Axis-Mediated Bioenergetic Functions.

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma viral oncogene homolog (KRAS) with p110α in lipid rafts with the use of naringenin (NGN), a citrus flavonoid, causes lipid raft-associated phosphatidylinositol 3-kinase (PI3K)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-protein kinase B (Akt)-regulated metabolic dysfunction of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), leading to apoptosis in human nasopharyngeal carcinoma (NPC) cells. The use of lethal-7g (let-7g) mimic and let-7g inhibitor confirmed that elevated let-7g resulted in a decrease in KRAS expression, which attenuated the PI3K-Rac1-Akt-BCL-2/BCL-xL-modulated mitochondrial energy metabolic functions. Increased let-7g depends on the suppression of the RNA-specificity of monocyte chemoattractant protein-induced protein-1 (MCPIP1) ribonuclease since NGN specifically blocks the degradation of pre-let-7g by NPC cell-derived immunoprecipitated MCPIP1. Converging lines of evidence indicate that the inhibition of MCPIP1 by NGN leads to let-7g upregulation, suppressing oncogenic KRAS-modulated PI3K-Rac1-Akt signaling and thereby impeding the metabolic activities of aerobic glycolysis and mitochondrial OXPHOS.

Correlation between KRAS Mutation and CTLA-4 mRNA Expression in Circulating Tumour Cells: Clinical Implications in Colorectal Cancer.

Combination strategies of KRAS inhibition with immunotherapy in treating advanced or recurrent colorectal carcinoma (CRC) may need to be assessed in circulating tumour cells (CTCs) to achieve better clinical outcomes. This study aimed to investigate the genomic variations of KRAS in CTCs and matched CRC tissues and compared mRNA expression of KRAS and CTLA-4 between wild-type and KRAS-mutated CTCs and CRC tissues. Clinicopathological correlations were also compared. Six known mutations of KRAS were identified at both codon 12 and codon 13 (c.35G>T/G12V, c.35G>A7/G12D, c.35G>C/G12A, c.34G>A/G12S, c.38G>C/G13A, and c.38G>A/G13D). Three CTC samples harboured the identified mutations (16.7%; 3/18), while fifteen matched primary tumour tissues (65.2%, 15/23) showed the mutations. CTCs harbouring the KRAS variant were different from matched CRC tissue. All the mutations were heterozygous. Though insignificant, CTLA-4 mRNA expression was higher in patients carrying KRAS mutations. Patients harbouring KRAS mutations in CTCs were more likely to have poorly differentiated tumours (p = 0.039) and with lymph node metastasis (p = 0.027) and perineural invasion (p = 0.014). KRAS mutations in CTCs were also significantly correlated with overall pathological stages (p = 0.027). These findings imply the genetic basis of KRAS with immunotherapeutic target molecules based on a real-time platform. This study also suggests the highly heterogeneous nature of cancer cells, which may facilitate the assessment of clonal dynamics across a single patient's disease.

Effects of DSPP and MMP20 Silencing on Key Signaling Pathways in Oral Squamous Cell Carcinoma Cells

Introduction: Oral carcinogenesis is a multistage process, featuring genetic and molecular alterations leading to rapid cell division, invasion, metastasis, and increased cell survival. Many of these alterations are due to perturbations in the cell signaling networks, which in turn lead to constitutive deregulation of the proteins involved in the regulatory pathways. Our recent reports show that the silencing of dentin sialophosphoprotein (DSPP) and its cognate matrix metalloproteinases 20 (MMP20) alters key tumorigenic hallmarks of oral squamous cell carcinoma (OSCC). Objective: This study, intended to advance our recent findings, focuses on determining the effects of silencing DSPP and its cognate MMP20 on the signaling pathways that control cell proliferation, differentiation, invasion and metastasis. Materials and Methods: DSPP and MMP20 were silenced individually and in combination, using adenovirus-mediated short hairpin RNA (shRNA) in OSCC cell line, OSC2, and the effects of silencing on the following pathways: EFGR; RAS-RAF; MEK; MAPK; ERK; JNK; NF-kB; TGFβ; and GSK3β, were analysed by western blot. Results: DSPP and MMP20 silencing decreased EGFR, KRAS, MEK1/2, MAPK, ERK, MEEK1, JNK, CREBP, p300, NF-kB,TGF β, SMAD7, GSK3 β, and β-catenin expressions. In contrast, the expression of IKKα and SMAD4 were increased in DSPP/MMP20-silenced group, compared with control group. Furthermore, DSPP-silencing alone was more effective than MMP20, or combined DSPP-MM20 silencing, in altering the levels of key proteins of each signaling pathway investigated. Conclusion: Our findings provide the basis for further studies aimed at verifying the effects of these alterations in the profiles of these proteins on the various hallmarks of oral carcinogenesis, and for understanding the molecular role of DSPP and MMP20 in OSCC. This is with a view to evaluating their diagnostic and prognostic utility as well as the values of DSPP/MMP20 as potential targets for design of chemotherapeutic agents for the treatment of OSCC patients.

Identification and gene expression analysis of circulating tumor cells with high expression of KRAS in pancreatic cancer

This study aims to establish a highly sensitive and specific method for single cell screening and identification of KRAS+/epithelial cell adhesion molecules (EpCAMs)/vimentin+/pancreatic cancer circulating tumor cells (CTCs) and to explore the clinical implications in the diagnosis of pancreatic cancer. A single cell separation and identification system for pancreatic cancer CTCs was constructed based on the preparation of KRAS immunolipid magnetic spheres. The function of the system was evaluated, the physical parameters of the immunoliposomes (IMLs) were characterized, and the isolation effect of the IMLs on CTCs was studied and compared with EpCAM and vimentin phenotypic CTCs. CTCs in the peripheral blood of 50 patients with pancreatic cancer confirmed by preoperative CT diagnosis and postoperative pathology were isolated and identified. The isolated CTCs were stained with CK immune antibody, observed, and identified, the number of CTCs was counted, and the relationship between the CTC count and the clinical malignant grade markers and grade indicators was examined. The consistency of the KRAS mutation rate between CTCs and tissues was analyzed and compared. It was found that the circulating tumor cell sorting system with a high expression of KRAS-ML could effectively enrich the circulating tumor cells with a positive expression of KRAS in pancreatic cancer. The pancreatic cancer CTC sorting and single-cell gene detection system can be used as a new detection technology of peripheral blood CTCs in patients with pancreatic cancer. As a minimally invasive liquid biopsy method, this technology has significant clinical practice implications for pancreatic cancer detection and effectiveness assessment.

Elimination of oncogenic KRAS in genetic mouse models eradicates pancreatic cancer by inducing FAS-dependent apoptosis by CD8+ T cells

Oncogenic KRASG12D (KRAS∗) is critical for the initiation and maintenance of pancreatic ductal adenocarcinoma (PDAC) and is a known repressor of tumor immunity. Conditional elimination of KRAS∗ in genetic mouse models of PDAC leads to the reactivation of FAS, CD8+ Tcell-mediated apoptosis, and complete eradication of tumors. KRAS∗ elimination recruits activated CD4+ and CD8+ Tcells and promotes the activation of antigen-presenting cells. Mechanistically, KRAS∗-mediated immune evasion involves the epigenetic regulation of Fas death receptor in cancer cells, via methylation of its promoter region. Furthermore, analysis of human RNA sequencing identifies that high KRAS expression in PDAC tumors shows a lower proportion of CD8+ Tcells and demonstrates shorter survival compared with tumors with low KRAS expression. This study highlights the role of CD8+ Tcells in the eradication of PDAC following KRAS∗ elimination and provides a rationale for the combination of KRAS∗ targeting with immunotherapy to control PDAC.

Microwave assists synthesis of spirothiazolidine derivatives to induce cervical cancer cell death-mediated apoptosis in vitro

Herein, a new series of spirothiazolidine derivatives 1–14 are effectively produced using microwave irradiation in a quick and efficient manner. IR, NMR, and mass spectra were used to characterize all new formed substances. Newly created substances based on spirothiazolidinone ring system were tested for their anticancer effects on the common human cervical cancer cell lines HEp-2 and HeLa to produce effective initiative anti-cervical cancer analogs in order to complement or replace the existing anti-cervical cancer chemicals. Among the suggested compounds, compounds 7, 9d, 12, and 13 were found to be the most effective. In HeLa and HEp-2 cells, compounds 7 and 13 significantly increased the expression of CAS3 and Bax while significantly decreasing the expression of KRAS, NRAS, and Bcl-2. We are able to conclude that Compounds 7 and 13 inhibited the RAS gene in order to cause apoptosis.

Targeting the Mutant Region in K-RAS by RNA Interference Controlling Cell Proliferation and Programmed Cell Death in Liver Cancer Cells.

Background Hepatocellular carcinoma (HCC) is a high-incidence and significant cause of cancer globally. HCC is the world's third most common death-related cancer and the sixth most common tumor. In HCC, various cellular signaling is activated to ensure malignancy transformation, angiogenesis, and metastasis. The most efficient signaling pathway in cancer is mitogen-activated protein kinase (MAPK), which controls malignancy and regulates apoptosis. The mutant k-ras gene led to the activation of the RAS protein with GTPase activity, which stimulated hepatocellular proliferation and transformation. Here we aim to investigate the correlation between the knock-down of k-ras and HCC evaluation in-vitro. &#x0D; Material and Methods We used the HepG2 cell line to investigate the direct connection between the k-ras expression profile and RAF/MEK pathway using a respective siRNA antagonist k-ras. &#x0D; Results Interestingly, the siRNA antagonist k-ras altered cell morphology and a number of the living HepG2 cells. In addition, the transfection of HepG2 cells with the firstly designed siRNA successfully reduced the expression profile k-ras and the relative gene expression of Raf-1 and Mek1, the downstream targets of RAS signaling. Furthermore, interleukin 8 (IL-8) and interleukin 6 (IL-6) were monitored in the fluids media at different time points following transfection. Both IL-6 and Il-8 production significantly increased in cells transfected with siRNA targeting k-ras. &#x0D; Conclusion Our findings provide evidence for the influential role of the k-ras mutated gene in HCC development and suggest the possible regulation of this gene as a potential treatment for HCC.

Spermine-Based Poly(β-amino ester)s for siRNA Delivery against Mutated KRAS in Lung Cancer.

Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(β-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(β-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(β-amino ester)s are promising materials for therapeutic siRNA delivery.