KRAS Protein Expression Research Articles

Abstract 5064: Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung cancer

Abstract Introduction: Oncogenic KRAS mutations define subsets of non-small cell lung cancer (NSCLC) with unique clinicopathologic and genomic features, and the recent development of direct KRAS inhibitors has changed the treatment landscape of these patients (pts). However, whether KRAS amplification is oncogenic and defines unique subset of potentially targetable NSCLCs is currently unknown. Methods: Clinicopathologic and genomic data were abstracted from multiple independent cohorts of NSCLCs which underwent tumor genomic profiling at the Dana-Farber Cancer Institute (Cohort 1), AACR-GENIE v.13, TCGA, and other 212 studies (Cohort 2). Whole transcriptome sequencing and quantitative proteomics data from the The Cancer Genome Atlas (TCGA) and the Cancer Cell Lines Encyclopedia (CCLE) were used to correlate KRAS amplification with KRAS RNA levels and protein expression. KRAS doxycycline-inducible models were generated to determine in vitro and in vivo oncogenicity of wild-type KRAS overexpression. Results: Among 15,341 pts with NSCLC identified, KRAS amplification was identified in 355 (2.4%) cases. Compared to KRAS not amplified cases, pts with KRAS amplified NSCLC were more likely to be males (52.4% vs 45.6%, p=0.01), have a history of smoking (82.1% vs 64.2%, p<0.01), and higher median pack-years (35 vs 23.5, p<0.01). KRAS amplification was also associated with higher median aneuploidy levels (as fraction of genome altered) (30.4% vs 14.3%, p<0.01), tumor mutational burden (p<0.01), and increased median PD-L1 expression (20% vs 5%, p=0.01). KRAS amplified cases were enriched with KRAS, POLE2, SLC34A2 and PALB2 mutations, while EGFR mutations were mutually exclusive (Q<0.1). Among NSCLC samples with transcriptomic and proteomic profiling from the TCGA and CCLE cohorts, KRAS amplification was associated with significantly increased KRAS mRNA (p<0.01) and protein expression (p<0.01), compared to KRAS diploid samples. Among pts with clinical outcomes data available (N=9,335), median overall survival (OS) from the date of diagnosis was significantly shorter in pts with KRAS amplified vs KRAS diploid genotype in both KRAS mutant (HR: 0.71, P=0.02) and KRAS WT (HR: 0.74, P=0.02) NSCLCs. In KRAS doxycycline-inducible models (BEAS-2B cells) we demonstrated increased cell proliferation and MAPK activation upon doxycycline-induced KRAS expression. In vivo, sustained expression of KRAS in animals constantly fed with doxycycline-diet triggered tumor formation and development of metastasis, indicating that WT KRAS overexpression is oncogenic. Conclusion: KRAS amplification defines a novel subset of NSCLCs characterized by distinct clinicopathologic and genomic features, and worse survival. Developing novel therapeutic strategies to target KRAS amplification may improve outcomes in pts with NSCLC. Citation Format: Biagio Ricciuti, Sandra V. Michelina, Joao Alessi, Xinan Wang, Federica Pecci, Alessandro Di Federico, Malini Gandhi, Giuseppe Lamberti, Enrico Patrucco, Lynette Sholl, Andrew Aguirre, Chiara Ambrogio, Mark Magdi Awad. Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5064.

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.

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.

Clinical, pathologic, and genomic hallmarks of KRAS-amplified non-small cell lung cancer.

9118 Background: KRAS-mutant non-small cell lung cancers (NSCLCs) have unique clinicopathologic and genomic features, and novel therapies are in development to target many KRAS-mutant tumor types. Whether KRAS amplification also defines a unique, potentially targetable, molecular subset of NSCLC is currently unknown. Methods: Clinicopathologic and genomic data were abstracted from multiple independent cohorts of pts whose cancers underwent tumor genomic profiling at the Dana-Farber Cancer Institute (Cohort 1), AACR Project GENIE v.13, The Cancer Genome Atlas (TCGA), and 212 other studies (Cohort 2). Patient (pt) outcomes were examined according to KRAS amplification status ( KRAS amplified vs KRAS non-amplified). Whole transcriptome sequencing and quantitative proteomics data from TCGA and the Cancer Cell Line Encyclopedia (CCLE) were used to correlate KRAS amplification with KRAS RNA levels and protein expression. Results: Among 15,341 pts with NSCLC, KRAS amplification was identified in 355 (2.3%) cases (median number of copies [range]: 11 [7-87]); the prevalence was similar in adenocarcinomas and squamous cell carcinomas. Compared to KRAS non-amplified cases, pts with KRAS-amplified NSCLC were more likely to be men (52.4% vs 45.6%, p = 0.01), have a history of smoking (82.1% vs 64.2%, p &lt; 0.01), and higher median pack-years (35 vs 23.5, p &lt; 0.01). However, median age was similar between the two groups (66 vs 66, p = 0.88). KRAS amplification was also associated with higher aneuploidy (median fraction of genome altered 30.4% vs 14.3%, p &lt; 0.01), tumor mutational burden (p &lt; 0.01 across different platforms), and increased median PD-L1 expression (20% vs 5%, p = 0.01). Of the 355 NSCLC samples with KRAS amplification, 152 (43%) had no concurrent oncogene driver mutations. In unbiased mutation enrichment analyses, KRAS-amplified tumors were enriched in concurrent mutations in KRAS, PALB2, POLE, and SLC34A2 (q &lt; 0.1), while KRAS non-amplified tumors were enriched for oncogenic EGFR mutations (q &lt; 0.1). Transcriptomic and proteomic profiling from the TCGA and CCLE cohorts demonstrated that KRAS amplification was associated with significantly increased KRAS mRNA (p &lt; 0.01) and protein expression (p &lt; 0.01), compared to KRAS non-amplified samples. In pts with available clinical outcomes data in the combined cohort (N = 9,335), the median overall survival (OS) from the date of diagnosis was significantly shorter in KRAS-amplified vs KRAS non-amplified cases (adjusted HR: 1.40, p &lt; 0.01). KRAS amplification was confirmed to confer significantly worse survival outcomes in pts with both KRAS wild-type (N = 6,695, adjusted HR 1.37, p = 0.02) and KRAS-mutant (N = 2,640, adjusted HR 1.34, p = 0.02) NSCLC. These results were independently replicated in Cohort 1 and Cohort 2. Conclusions: KRAS amplification defines a novel molecular subset of NSCLC characterized by distinct clinicopathologic and genomic features and worse survival.

Abstract B014: Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS

Abstract Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene detected in 30% of all human cancers. Drugs that can impede the function of oncogenic KRAS offer exceptional therapeutic value. However, direct targeting KRAS is challenging due to its protein structure that lacks deep binding pockets for small-molecule inhibitors. Despite the recent success in targeting the G12C allele, targeted therapy for another hotspot mutant – G12V mutant KRAS has not been described. Here, we utilize CRISPR-Cas9 based genome-wide knockout screens and leverage a high-throughput microfluidic cell sorting platform to identify genes that regulate mutant KRAS harboring G12V mutation. The screens were conducted in two colorectal cancer cell lines HT29 and SW480, expressing wild-type KRAS and homozygous G12V KRAS, respectively. We have identified a list of genes whose loss-of-function reduces wild-type or mutant G12V KRAS protein expression. Our top gene identified from the mutant KRAS screen - denoted as selective-mutant KRAS modulator (SMKM) has shown promising results in selectively decreasing G12V KRAS protein expression. Through multiple assays including live cell imaging, western blot, flow cytometry, and proteomic analysis in SMKM knockout cells and SMKM inhibitor-treated cells. We found highly selective reduction in G12V KRAS protein expression. More importantly, genetically or pharmacologically inhibiting SMKM has demonstrated anti-tumor activity. We conducted cell viability assays including CellTiter-glo, MTT, and clonogenic assay. We treated a panel of G12V KRAS mutant and wild-type KRAS cell lines with SMKM inhibitor and found significantly reduced cell viability in G12V KRAS mutant cell lines across colorectal and lung cancer. To further demonstrate the downstream impact on mutant KRAS signaling, secondary assays including western blot and Lumit immunoassay were conducted. We observe a reduction in the aberrant phospho-ERK and phospho-AKT signaling in SMKM inhibitor treated cells. Our in vivo efficacy trial using heterozygous G12V KRAS lung cancer NCI-H441 xenograft model has shown SMKM inhibition significantly reduces tumor growth and increases the survival rate. Together, these data indicate the feasibility of selectively targeting G12V KRAS with SMKM inhibition. Lastly, our results provide a first-in-class small molecule oral inhibitor based selective protein clearance as an effective anti-G12V mutant KRAS therapeutic strategy. Citation Format: Xiyue Hu, Randy Singh, Shana Kelley. Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B014.

KRAS Protein Expression in Oral Squamous Cell Carcinoma: A Potential Marker for Progression and Prognosis.

Emerging evidence suggests that KRAS could play an important role in squamous cell carcinoma; however, its role in oral squamous cell carcinoma (OSCC) is largely unknown. The aim of the current study was to investigate the expression of KRAS, Ki-67, Cyclin D1, and Bcl2 in OSCC and their association with clinicopathological features. Forty paraffin blocks of retrospective histologically diagnosed cases of OSCC and 20 blocks of oral leukoplakia with epithelial dysplasia were obtained from two hospitals between 2018 and 2021. The paraffin-embedded tissue was analyzed for the expression of KRAS for oral epithelial dysplasia and OSCC, and ki-67, Cyclin D1, and bcl2 were analyzed only for OSCC. The results were correlated with each other and with different clinicopathological features and were statistically analyzed. KRAS expression was significantly associated with histological tumor grade, tumor extent, presence of nodal and distant metastasis, pathological stage, and the presence of lymphovascular invasion (P=<0.001, 0.001, 0.001, 0.009, <0.001, and <0.001, respectively). The KRAS expression was positively correlated with the histological grade, tumor extent, nodal status, and the pathological stage (r=0.712, 0.649, 0.646, and 0.865, respectively). A positive correlation was also found with the expression of Bcl2, Cyclin D1, and Ki-67 (r=0.81, 0.723, and 0.698, respectively). The KRAS expression in oral epithelial dysplasia was significantly lower than that in OSCC (P=0.003). KRAS may be a potential prognostic marker for OSCC and may play a role in its progression.

KRAS protein expression becomes progressively restricted during embryogenesis and in adulthood.

KRAS mutants are common in many cancers and wild-type KRAS is essential in development as its absence causes embryonic lethality. Despite this critical role in development and disease, the normal expression pattern of KRAS protein is still largely unknown at the tissue level due to the lack of valid antibodies. To address this issue, we used the citrine-Kras mouse model in which the Citrine-KRAS (Cit-K) fusion protein functions as a validated surrogate of endogenous KRAS protein that can be detected on tissue sections by immunolabeling with a GFP antibody. In the embryo, we found expression of KRAS protein in a wide range of organs and tissues. This expression tends to decrease near birth, mainly in mesenchymal cells. During transition to the adult stage, the dynamics of KRAS protein expression vary among organs and detection of KRAS becomes restricted to specific cell types. Furthermore, we found that steady state KRAS protein expression is detectable at the cell membrane and in the cytoplasm and that this subcellular partitioning differed among cell types. Our results reveal hitherto unanticipated dynamics in developmental, tissular, cell-specific and subcellular expression of KRAS protein. They provide insight into the reason why specific cell-types are sensitive to KRAS mutations during cancer initiation.

Targeting GRP78 suppresses oncogenic KRAS protein expression and reduces viability of cancer cells bearing various KRAS mutations

KRAS is the most commonly mutated oncogene in human cancers with limited therapeutic options, thus there is a critical need to identify novel targets and inhibiting agents. The 78-kDa glucose-regulated protein GRP78, which is upregulated in KRAS cancers, is an essential chaperone and the master regulator of the unfolded protein response (UPR). Following up on our recent discoveries that GRP78 haploinsufficiency suppresses both KRASG12D-driven pancreatic and lung tumorigenesis, we seek to determine the underlying mechanisms. Here, we report that knockdown of GRP78 via siRNA reduced oncogenic KRAS protein level in human lung, colon, and pancreatic cancer cells bearing various KRAS mutations. This effect was at the post-transcriptional level and is independent of proteasomal degradation or autophagy. Moreover, targeting GRP78 via small molecule inhibitors such as HA15 and YUM70 with anti-cancer activities while sparing normal cells significantly suppressed oncogenic KRAS expression in vitro and in vivo, associating with onset of apoptosis and loss of viability in cancer cells bearing various KRAS mutations. Collectively, our studies reveal that GRP78 is a previously unidentified regulator of oncogenic KRAS expression, and, as such, augments the other anti-cancer activities of GRP78 small molecule inhibitors to potentially achieve general, long-term suppression of mutant KRAS-driven tumorigenesis.

KRAS Promoter Methylation Status and miR-18a-3p and miR-143 Expression in Patients With Wild-type KRAS Gene in Colorectal Cancer.

Although some mutations of KRAS proto-oncogene, GTPase (KRAS) have been associated with the prognosis and therapeutic management of colorectal cancer (CRC), the epigenetic mechanisms (DNA methylation and microRNA expression) that regulate wild-type KRAS expression in patients with CRC are poorly known. The aim of this study was to establish whether there is a relationship between the expression of the wild-type KRAS gene, the methylation status of its distal promoter, and miR-143 and miR-18a-3p levels in samples of sporadic CRC. A total of 51 cases of sporadic CRC with wild-type KRAS were analyzed. The expression levels of KRAS mRNA, miR-18a-3p, miR-143, and KRAS protein, as well as methylation in the distal promoter of the KRAS gene were evaluated. In the analyzed cases, KRAS mRNA expression was detected in 51.1%; wild-type KRAS protein was found in the membrane in 31.4% and in the cytoplasm in 98% of cases. An inverse relationship of marginal significance was observed between miR-18a-3p and KRAS protein expression in the cytoplasm (odds ratio=0.14, 95% confidence interval=0.012-1.092; p=0.08). The methylation status of the distal promoter of KRAS at four CpG islands was analyzed in 30 cases (58.8%): partial methylation of the four CpG islands evaluated was observed in two cases (6.7%). In these cases, KRAS protein expression was not evidenced at the membrane level; miR-18a-3p expression was not detected either but high expression of miR-143 was observed. No association was found between the expression levels of KRAS mRNA, miR-18a-3p, miR-143 and methylation status. Methylation status was detected with low frequency, thus being the first report of methylation in wild-type KRAS.

Immunohistochemical expression of K-ras protein and p53 in chronic pancreatitis and precursor lesions of pancreatic carcinoma- 5 year study in a tertiary care centre

K-ras mutation is an early event and p53 mutation is a late event in the tumorigenesis pathway of carcinoma associated with Chronic Pancreatitis. To identify the immunohistochemical expression of K-ras protein and p53 in surgical specimens of pancreas with chronic pancreatitis.: A 5 year study of chronic pancreatitis with associated pancreatic lesions received in the Histopathology Department, Government Medical College, Trivandrum. All cases of chronic pancreatitis, (n=60) were histopathologically examined along with the adjacent pancreatic tissue which showed precursor lesions. Immunohistochemical expression of K-ras protein and p53 in all cases were studied. In a total 60 cases of chronic pancreatitis, adjacent pancreatic tissue showed ductal adenocarcinoma in 20 cases (30%). Pancreatic Intraepithelial neoplasm (PanIN) was present in 6 cases accounting for 10%. 1 case was IPMN and one case was associated with serous cystadenoma. 7% of chronic pancreatitis showed K-ras mutations. Immunostaining for mutated p53 protein always was negative. p53 was positive in Pan IN 1A , Pan IN 1B, IPMN and carcinoma cells in 0 of 4(0%), 0 of 2(0%), 0 of 1(0%), and 18 of 20(90%), respectively, and K-ras was positive in 3 of 4 (75%), 2 of 2(100%), 1of 1(100%) and 12 of 20(60%), cases respectively. Chronic pancreatitis and precursor lesions may progress to the development of pancreatic cancer. Further molecular studies for evaluation of oncogenes may be done in future for targeted therapy in pancreatic cancer.

Activating KRAS and GNAS mutations in heterotopic submucosal glands of the stomach.

The heterotopic submucosal gland (HSG) is a common incidental finding in gastrectomy specimens. The majority of HSGs are small incidental lesions, which are also known as gastritis cystica profunda. However, larger lesions may appear as an inverted growth of well-organized mucosa referred to as gastric inverted polyps. To determine whether genetic alterations are involved in HSG development, we analyzed 63 gastric HSG lesions using targeted next-generation sequencing and immunohistochemistry. Histologically, HSG lesions consistently had areas of pyloric gland differentiation with variable extent of foveolar differentiation. Although the background mucosa showed intestinal metaplasia in most cases (98%), intestinal-type epithelium was seen in only one HSG lesion (2%). Sequencing analysis identified activating KRAS, BRAF, CTNNB1, and GNAS mutations in 34 (54%), 1 (2%), 1 (2%), and 7 (11%) lesions, respectively. HSG lesions harboring a KRAS mutation were more likely to present extensive foveolar differentiation (P = 0.013) and absence of parietal cells (P = 0.0081). Five HSG lesions had a dysplastic component, and concordant genetic alterations were detected between the non-dysplastic and dysplastic areas of two lesions that were successfully analyzed. Immunohistochemical staining demonstrated diffuse expression of mutant KRAS protein in lesions with the most common genetic alteration, KRAS G12D. Our study demonstrated that a major proportion of HSGs were proliferative lesions associated with oncogenic mutations, with more than half of lesions harboring activating KRAS mutations.

The development of cigarette smoke induced chronic pancreatitis in mice is associated with increased expression of K-Ras and NF-κB.

Epidemiological studies have demonstrated a strong association between cigarette smoking (CS) and chronic pancreatitis (CP); however, the exact mechanisms of this phenomenon remains unknown. The authors have previously shown that increased Ras expression activates the NF-κB mediated pathway and promotes development of CP. However, it is unclear whether a similar phenomenon occurs in CS-induced CP. Therefore, the aim of the study was to determine whether CS increases the expression of K-Ras, and promotes the development of CP in mice exposed to repeated episodes of acute pancreatitis (AP). C57BL6/cmdb mice were exposed to CS or a sham treatment for 12 weeks. After one week of exposure, half of the animals from both groups were additionally subjected to repeated cerulein treatment (once a week, for 10 consecutive weeks) to mimic recurrent episodes of AP. Extension of pancreatic damage was determined histologically by H&E and Trichrome staining. The expression of K-Ras protein and downstream components (NF-κB, Cox-2, TGF-β) was evaluated by immunohistochemistry. C57BL6/cmdb mice exposed to CS or cerulein alone did not develop any chronic pancreatic damage. However, concomitant treatment with both of these agents caused focal acinar atrophy, with slight intralobular and perivascular areas of fibrosis, and inflammatory cells infiltration resembling mild CP. Moreover, immunohistochemistry examinations revealed increased pancreatic expression of K-Ras and NF-κB only in mice treated both with CS and cerulein. CS promotes development of CP in mice exposed to repeated episodes of AP. This process may be, at least partially, related to increased expression of K-Ras and NF-κB protein.

LncRNA IGFL2-AS1 Promotes the Proliferation, Migration, and Invasion of Colon Cancer Cells and is Associated with Patient Prognosis.

BackgroundLncRNAs play an important role in tumor initiation and development. However, the underlying involvement of lncRNA expression in colorectal carcinoma remains to be clarified.MethodsAll analyses were performed in R software v4.0, SPSS v13.0, and GraphPad Prism 8. The “limma” package was used to identify differentially expressed lncRNAs between two groups with the threshold of |logFC| >1 and P <0.05. The “Survival” package was used to conduct survival analysis. HCT8 and SE480 cell lines were used to conduct further phenotype experiments, including transwell, wound-healing, CCK8 and colony formation assay. Gene set enrichment analysis was used to explore the biological pathway difference in high and low IGFL2-AS1 patients.ResultsThe lncRNA IGFL2-AS1 was highly expressed in colon adenocarcinoma (COAD) tissue and cell lines (HCT116, HCT8, HCT129, and SW480). The COAD patients with high IGFL2-AS1 were associated with a worse prognosis. Meanwhile, the knockdown of IGFL2-AS1 could significantly suppress the proliferation and invasion of COAD cells. Gene set enrichment analysis showed that the top five biological pathways involving IGFL2-AS1 were angiogenesis, epithelial–mesenchymal transition, KRAS signaling, myogenesis, and coagulation. Western blot results showed that the inhibition of IGFL2-AS1 could significantly reduce the N-cadherin, HIF1A and KRAS protein expression, yet increase the E-cadherin protein level. IGFL2-AS1 was also positively correlated with M0 macrophages, M2 macrophages, and neutrophils but negatively correlated with CD4+ memory T cells and CD8+ T cells.ConclusionIGFL1-AS1 could seriously worsen patient outcomes and facilitate COAD progression, thus serving as an independent tumor marker.

PBDEs affect inflammatory and oncosuppressive mechanisms via the EZH2 methyltransferase in airway epithelial cells

AimsWe aimed to investigate the effect of PBDEs (47, 99, 209) on cellular events involved in epigenetic modification, inflammation, and epithelial mesenchymal transition (EMT). Materials and methodsWe studied: 1) ERK1/2 phosphorylation; 2) Enhancer of Zester Homolog 2 (EZH2); 3) Histone H3 tri-methylated in lysine 27 (H3K27me3); 4) K-RAS; 5) silencing disabled homolog 2-interacting protein gene (DAB2IP), 6) let-7a; 7) Muc5AC/Muc5B, and 8) IL-8 in a 3D in vitro model of epithelium obtained with primary Normal Human Bronchial Epithelial cells (pNHBEs) or A549 cell line, chronically exposed to PBDEs (47, 99, 209). Key findingsPBDEs (10 nM, 100 nM and 1 μM) increased ERK1/2 phosphorylation, and EZH2, H3K27me3, and K-RAS protein expression, while decreased DAB2IP and Let-7a transcripts in pNHBEs ALI culture. Furthermore PBDEs (47, 99) (100 nM) increased Muc5AC and Muc5B mRNA, and PBDE 47 (100 nM) IL-8 mRNA via EZH2 in pNHBEs. Finally, PBDEs (100 nM) affected EZH2, H3K27me3, K-RAS protein expression, and DAB2IP, Let-7a transcripts and cell invasion in A549 cells. Gsk343 (methyltransferase EZH2 inhibitor) (1 mM) and U0126 (inhibitor of MEK1/2) (10 μM) were used to show the specific effect of PBDEs. SignificancePBDE inhalation might promote inflammation/cancer via EZH2 methyltransferase activity and H3K27me3, k-RAS and ERk1/2 involvement, generating adverse health outcomes of the human lung.

Regulation of KRAS protein expression by miR-544a and KRAS-LCS6 polymorphism in wild-type KRAS sporadic colon adenocarcinoma.

Colorectal carcinoma (CRC) results from the accumulation of genetic mutations and alterations in signaling pathways. KRAS is mutated in 40% of CRC cases and is involved in increased tumor cells proliferation and survival. Although KRAS mutations are a dominant event in CRC tumorigenesis, increased wild-type KRAS expression has a similar effect on accelerated tumor growth. In this study, we investigated the KRAS status in correlation with clinicopathological features in sporadic CRC and more importantly the role of let-7a-5p and miR-544a-3p in the regulation of wild-type KRAS protein expression in the tumor center (T1) and invasive tumor front (T2). Analysis showed that 39.1% of tumor samples had KRAS mutations. In wild-type KRAS tumors, 62.0% were positive for KRAS protein expression and there was a higher percentage of KRAS-positive tumor cells and a higher intensity of immunohistochemical reaction in T2 than in T1 samples. This could not be attributed to differences in KRAS mRNA levels, suggesting regulation via miR-544a-3p expression which was significantly decreased in T2 samples. Furthermore, we demonstrated that tumor samples carrying the KRAS-LCS6 variant allele had significantly higher protein expression of the wild-type KRAS. Our results suggest the role of the KRAS-LCS6 polymorphism and miR-544a-3p expression in the regulation of wild-type KRAS protein expression in sporadic CRC.

Potential Role of KRAS and BRAF in Epithelial Ovarian Cancer

KRAS and BRAF are key proto-oncogenes of MAPK/ERK pathway and are essential for cell growth and proliferation. Mutations of these oncogenes are known to have role in pathogenesis of epithelial ovarian carcinoma (EOC). However, limited studies have evaluated the role of KRAS and BRAF protein expression. Hence, this study aimed to explore their role in EOC patients, specifically to correlate KRAS and BRAF protein expression with the histological subtypes and other histopathological prognostic factors. Immunohistochemistry was performed using paraffin-embedded tumor tissue blocks of 55 untreated and histologically confirmed EOC patients and 20 benign ovarian tumors to study KRAS and BRAF protein expression. Data were analyzed using SPSS software. Cytoplasmic expression of both KRAS and BRAF was observed in EOC and benign tumors. KRAS protein expression was significantly higher in serous EOC patients than the mucinous EOC patients (P = 0.016). KRAS expression was also significantly positively correlated with the presence of lymphatic permeation (P = 0.030), while a trend toward significance was found with vascular permeation (P = 0.052). Furthermore, BRAF protein expression was significantly higher in EOC patients than in benign ovarian tumors (P = 0.027). Its expression was significantly higher in patients with moderately/poorly differentiated tumors than well-differentiated tumors (P = 0.008), and in patients having serous EOC than in mucinous EOC (P = 0.001). A trend of higher BRAF expression was also observed in patients with lymphnode metastasis (P = 0.079). KRAS and BRAF proteins have a potential role in EOCs. However, further studies in larger group of patients and follow-up for a longer period are required to identify their role in prognosis.

Targeted multiplex proteomics (TMP) and genomics of early-onset colorectal cancer (EO-CRC).

97 Background: The incidence and mortality of early-onset colorectal cancer (EO-CRC) is on the rise. Consequently, there is an urgent unmet need to better understand their unique tumor biology to expand therapeutic options and improve clinical outcomes. Methods: Exploratory Targeted multiplex proteomics (TMP) and targeted 648 gene panel was performed on specimens from 35 patients with resected colon cancer diagnosed at age &lt; = 40 years. TMP panel consisted of 72 proteins involved in differentiation, tumorigenesis, and response to chemotherapy, targeted therapy, and immunotherapy. Clinicopathologic and genomic data were also collected. Results: The median age of diagnosis was 33 years. The cohort included 15 male and 20 female patients. 20 (57%) had left-sided tumors and 6 (17%) had stage IV disease. Notable genomic alterations included mutations in: BRAF V600E (2/35); RAS (15/35); PIK3CA exon 9 or 20 (5/35); and ERBB2 (2/35). One patient exhibited ERBB2 amplification. 9/35 tumors were MSI-H. TMP analysis revealed overexpression of chemotherapy resistance proteins in several patients: ALDH1A1:16/35; ERCC1:1/35; GART:26/35; MDR1:9/35; MGMT:5/35; RRM1:6/35; TUBB3:1/35; TYMS:2/35; XRCC1:11/35. In contrast, some tumors exhibited elevated biomarkers of chemosensitivity: hENT1:8/35; DHFR:12/35; TYMP:15/35; OPRT:8/35; SLFN11:1/35; TLE:11/35; TOPO1:12/35; TOPO2A:1/35. Protein targets of cell signaling pathways were overexpressed in a number of tumors: CAT:16/35; CAV-1: 6/35; CBL:2/35; E-Cadherin: 19/35; HSP90A:16/35; HSP90B:18/35; MET:5/35; NQO1:18/35; paxillin:4/35; SRC:21/35; STAT3:11/35. Regarding EGFR and KRAS, none of the tumors exhibited elevated protein expression level. Furthermore, RAS mutational status did not correlate with the level of EGFR or KRAS protein expression. Antibody drug conjugate biomarkers were observed. HER2 overexpression was noted in one patient who had a confirmed ERBB2 amplification. Regarding immunotherapy targets, PDL-1 protein was not overexpressed in any tumor, whereas MSLN and TROP2 were elevated in 1/35 and 2/35 patients, respectively. Conclusions: TMP analysis of EO-CRC patients revealed marked heterogeneity in the expression of proteins involved in differentiation, tumorigenesis, and response to chemotherapy, targeted therapy, and immunotherapy. Differential protein expression may provide insight into therapeutic vulnerabilities for EO-CRC. Furthermore, the discordance between detected genomic alterations and protein expression levels highlights the complementary nature of genomic sequencing and TMP analysis.

Impact of K-Ras Over-expression in Laryngeal Squamous Cell Carcinoma

Oncogene up-regulation combined with suppressor gene down-regulation is a crucial genetic combination that promotes cell neoplastic phenotype and progressively malignant transformation in solid malignancies, including laryngeal squamous cell carcinoma (LSCC). Among oncogenes, the Kirsten ras oncogene homolog (K-Ras) is involved in LSCC onset and progression. Sixty (n=60) primary LSCC tissue sections were analyzed by immunohistochemistry (IHC). Digital image analysis (DIA) was also implemented for measuring K-Ras protein expression levels. High K-Ras protein expression levels were observed in 20/60 (33.3%) LSCC tissue sections, whereas the rest of the cases (n=40; 66.7%) demonstrated low expression. Overall K-Ras expression was borderline significantly associated to the grade of the examined malignancies (p=0.048), whereas no other strong statistical correlations were identified. A progressive K-Ras overexpression was observed in all grades of the examined cases. K-Ras over expression is correlated to a progressive dedifferentiation in LSCC.

Accuracy of endoscopic ultrasound-guided needle aspiration specimens for molecular diagnosis of non-small-cell lung carcinoma.

BACKGROUNDEndoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are highly sensitive for diagnosing and staging lung cancer. In recent years, targeted therapy has shown great significance in the treatment of non-small cell lung carcinoma (NSCLC). Using these minimally invasive techniques to obtain specimens for molecular testing will provide patients with a more convenient diagnostic approach.AIMTo evaluate the feasibility and accuracy of tissue samples obtained using EUS-FNA and EBUS-TBNA for molecular diagnosis of NSCLC.METHODSA total of 83 patients with NSCLC underwent molecular testing using tissues obtained from EUS-FNA or EBUS-TBNA at the Tianjin Medical University Cancer Hospital from January 2017 to June 2019. All enrolled patients underwent chest computed tomography or positron emission tomography/computed tomography prior to puncture. We detected abnormal expression of EGFR, KRAS, MET, HER2, ROS1 and anaplastic lymphoma kinase protein. Two patients failed to complete molecular testing due to insufficient tumor tissue. The clinical features, puncture records, molecular testing results and targeted treatment in the remaining 81 patients were summarized.RESULTSIn a total of 99 tissue samples obtained from 83 patients, molecular testing was successfully completed in 93 samples with a sample adequacy ratio of 93.9% (93/99). Biopsy samples from two patients failed to provide test results due to insufficient tumor tissue. In the remaining 81 patients, 62 cases (76.5%) were found to have adenocarcinoma, 11 cases (13.6%) had squamous cell carcinoma, 3 cases (3.7%) had adenosquamous carcinoma and 5 cases (6.2%) had NSCLC-not otherwise specified. The results of molecular testing showed EGFR mutations in 21 cases (25.9%), KRAS mutations in 9 cases (11.1%), ROS-1 rearrangement in 1 case (1.2%) and anaplastic lymphoma kinase-positive in 5 cases (6.2%). Twenty-four patients with positive results received targeted therapy. The total effectiveness rate of targeted therapy was 66.7% (16/24), and the disease control rate was 83.3% (20/24).CONCLUSIONTissue samples obtained by EUS-FNA or EBUS-TBNA are feasible for the molecular diagnosis of NSCLC and can provide reliable evidence for clinical diagnosis and treatment.