K-ras 4A Expression Research Articles

K-Ras4A Plays a More Significant Role than K-Ras4B in Ductal Carcinoma of Breast.

K-Ras mutations rarely occur in breast cancer. However, studies have supported that K-Ras upregulation is involved in breast cancer pathogenesis. Two main K-Ras transcript variants; K-Ras4A and K-Ras4B, originate from the alternative splicing of exon 4. In this study, we aimed to evaluate variations in the expression of K-Ras4A and K-Ras4B and their role in breast ductal carcinoma. Total RNA was extracted from breast tumors, and the NATs obtained via mastectomy. Patients were selected from new cases of breast cancer with no prior history of chemotherapy. Relative mRNA expression was calculated based on a pairwise comparison between the tumors and the NATs following normalization to the internal control gene. Predictive values of the transcript variants were examined by ROC curve analysis. A statistically significant increase was found in the K-Ras4A and K-Ras4B expression with the mean fold changes of 7.58 (p = 0.01) and 2.47 (p = 0.001), respectively. The K-Ras4A/K-Ras4B ratio was lower in the tumors than that of the normal tissues. ROC curve analysis revealed the potential of K-Ras4A (AUC: 0.769) and K-Ras4B (AUC: 0.688) in breast cancer prediction. There was also a significant association between K-Ras4B expression and HER2 statues (p = 0.04). Furthermore, a significant link was detected between K-Ras4A expression and pathological prognostic stages (p = 0.04). Our findings revealed that expression levels of K-Ras4A and K-Ras4B is higher in the tumor compared to the normal breast tissues. Increase in K-Ras4A expression was more significant than that of K-Ras4B.

The role of KRAS splice variants in cancer biology.

The three mammalian RAS genes (HRAS, NRAS and KRAS) encode four proteins that play central roles in cancer biology. Among them, KRAS is mutated more frequently in human cancer than any other oncogene. The pre-mRNA of KRAS is alternatively spliced to give rise to two products, KRAS4A and KRAS4B, which differ in the membrane targeting sequences at their respective C-termini. Notably, both KRAS4A and KRAS4B are oncogenic when KRAS is constitutively activated by mutation in exon 2 or 3. Whereas KRAS4B is the most studied oncoprotein, KRAS4A is understudied and until recently considered relatively unimportant. Emerging work has confirmed expression of KRAS4A in cancer and found non-overlapping functions of the splice variants. The most clearly demonstrated of these is direct regulation of hexokinase 1 by KRAS4A, suggesting that the metabolic vulnerabilities of KRAS-mutant tumors may be determined in part by the relative expression of the splice variants. The aim of this review is to address the most relevant characteristics and differential functions of the KRAS splice variants as they relate to cancer onset and progression.

KRAS4A induces metastatic lung adenocarcinomas in vivo in the absence of the KRAS4B isoform

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a KrasFSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras+/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras+/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences.

Abstract A13: KRAS4A directly regulates hexokinase 1

Abstract The KRAS oncogene is alternatively spliced, yielding KRAS4A and KRAS4B, which differ only in their membrane-targeting sequences. Tumors that harbor mutant KRAS manifest enhanced glucose utilization. We report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, establishing HK1 as an effector of KRAS4A. The interaction is unique to KRAS4A because the palmitoylation/depalmitoylation cycle of this RAS isoform permits colocalization with HK1 on the outer mitochondrial membrane. Our findings constitute the first demonstration of direct regulation of a metabolic enzyme by a RAS protein and the first biochemical distinction between the KRAS splice variants. KRAS4A expression in cancer may drive unique metabolic vulnerabilities that can be therapeutically exploited. Citation Format: James P. Mahaffety, Caroline R. Amendola, Wei-Ching Chen, Alec Kimmelman, Allan Balmain, Mark R. Philips. KRAS4A directly regulates hexokinase 1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A13.

KRAS4A directly regulates hexokinase 1.

The most frequently mutated oncogene in cancer is KRAS, which utilizes alternative fourth exons to generate two gene products, KRAS4A and KRAS4B, that differ only in their C-terminal membrane-targeting region 1. Because oncogenic mutations occur in exons 2 or 3, when KRAS is activated by mutation two constitutively active KRAS proteins are encoded, each capable of transforming cells 2. No functional distinctions among the splice variants have been established. Oncogenic KRAS alters tumor metabolism 3. Among these alterations is increased glucose uptake and glycolysis, even in the presence of abundant oxygen 4 (the Warburg Effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes 3–5, direct regulation of metabolic enzymes has not been examined. We report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, establishing HK1 as an effector of KRAS4A. The interaction is unique to KRAS4A because the palmitoylation/depalmitoylation cycle of this RAS isoform permits co-localization with HK1 on the outer mitochondrial membrane (OMM). KRAS4A expression in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.

A cross-sectional study examining the expression of splice variants K-RAS4A and K-RAS4B in advanced non-small-cell lung cancer patients

ObjectivesMammalian cells differently express 4 RAS isoforms: H-RAS, N-RAS, K-RAS4A and K-RAS4B, which are important in promoting oncogenic processes when mutated. In lung cancer, the K-RAS isoform is the most frequently altered RAS protein, being also a difficult therapeutic target. Interestingly, there are two K-RAS splice variants (K-RAS4A and K-RAS4B) and little is known about the role of K-RAS4A. Most studies targeting K-RAS, or analysing it as a prognostic factor, have not taken into account the two isoforms. Consequently, the in-depth investigation of them is needed. MethodsThe present study analysed 98 specimens from advanced non-small cell lung cancer (NSCLC) adenocarcinoma patients originated from Brazil. The alterations present in K-RAS at the DNA level (Sanger sequencing) as well as the expression of the splicing isoforms at the RNA (qRT-PCR) and protein levels (immunohistochemistry analysis), were evaluated. Possible associations between clinicopathological features and the molecular findings were also investigated. ResultsOur results showed that in the non-smoking population, the cancer incidence was higher among women. In contrast, in smokers and former smokers, the incidence was higher among men. Regarding sequencing results, 10.5% of valid samples presented mutations in exon 2, being all wild-type for exon 3, and the most frequently occurring base change was the transversion G → T. Our qRT-PCR and immunohistochemical analysis showed that both, K-RAS4A and K-RAS4B, were differently expressed in NSCLC tumour samples. For example, tumour specimens showed higher K-RAS4A mRNA expression in relation to commercial normal lung control than did K-RAS4B. In addition, K-RAS4B protein expression was frequently stronger than K-RAS4A in the patients analysed. ConclusionOur results highlight the differential expression of K-RAS4A and K-RAS4B in advanced adenocarcinoma NSCLC patients and underline the need to further clarify the enigma behind their biological significance in various cancer types, including NSCLC.

Roles of palmitoylation and the KIKK membrane-targeting motif in leukemogenesis by oncogenic KRAS4A.

BackgroundWe have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS-related cancers. For KRAS-driven cancer, although much research has been focused on the KRAS4B splice variant, which does not undergo palmitoylation, KRAS4A has recently been shown to play an essential role in the development of carcinogen-induced lung cancer in mice and to be widely expressed in human cancers. However, the role of palmitoylation in KRAS4A tumorigenesis is not clear.MethodsThe expression of KRAS4A in KRAS-mutated leukemia cell lines and acute myeloid leukemia (AML) cells were checked using western blotting and reverse transcriptions-quantitative polymerase chain reaction (RT-qPCR) analysis, respectively. The leukemogenic potentials of oncogenic KRAS4A and its palmitoylation-defective mutants were examined by a mouse bone marrow transduction and transplantation model and the in vitro transformation assays. The activation of the RAS downstream signaling pathways and the membrane localizations of the KRAS4A and its mutants were analyzed via western blot analysis and confocal microscopy, respectively.ResultsWe show here that KRAS4A is expressed in human leukemia cell lines and in AML cells harboring KRAS mutations and that mutation at the palmitoylation site of oncogenic KRAS4A significantly abrogates its leukemogenic potential. However, unlike NRAS, palmitoylation-defective KRAS4A still induces leukemia in mice, albeit with a much longer latency. Using NRAS/KRAS4A chimeric constructs, we found that the KIKK motif of KRAS4A contributes to the transforming activity of KRAS4A. Mutations at both palmitoylation site and the KIKK motif abolish the ability of oncogenic KRAS4A to induce leukemia in mice.ConclusionsOur studies suggest that therapies targeting RAS palmitoylation may also be effective in treating KRAS4A associated malignancies and that interfering the KIKK membrane-targeting motif would enhance the therapeutic effectiveness.

Abstract 1932: RNA-seq differential transcription analysis of TCGA colorectal cancer (CRC) transcriptomes reveals subtype-specific isoform usage

Abstract Background: Differential transcription analysis may reveal transcriptome aberrations associated with cancer progression and help identify important drivers in molecularly distinct subtypes of a disease. Methods: We developed a data-driven bioinformatics pipeline (DiffSplice) that performs differential transcription analysis at the alternative splicing (AS) level and allows accurate transcriptome reconstruction through joint analysis of all RNA-seq samples. In this study, we applied DiffSplice to RNA-seq data from TCGA CRC (n = 686) and evaluated the expression patterns of the detected AS events across the four consensus molecular subtypes (CMS) of the disease (n = 511): CMS1 - MSI Immune (hypermutated, n = 76); CMS2 - Canonical (epithelial, WNT/MYC activation, n = 220); CMS3 - Metabolic (epithelial, enriched for KRAS mutations, n = 72); and CMS4 - Mesenchymal (stromal invasion, TGFB activation, n = 143). Results: A total of 15,613 AS events were detected, including 5,128 events that involved novel splice junctions not cataloged in UCSC GAF2.0 transcriptome annotation. Exon skipping was the most common pattern with 6,482 occurrences, followed by 4,962 alternative splice sites, 2,689 alternative 5′/3′ transcription sites and 1,262 retained introns. In the subset of samples with CMS classification, a cross-subtype comparative analysis revealed 1,835 differentially transcribed loci from 1,634 genes, including 759 novel junctions (FDR<0.01). Of particular interest, we found that KRAS 4A expression levels were higher in CMS3 samples (p = 0.0070), in line with the known effect of this isoform as a negative regulator of mutant KRAS alleles. In addition, expression of the CD44v isoform, a marker of epithelial cancer stem cells, was higher in CMS2 and CMS3 samples (p = 0.015). Alternatively, CD44s isoform expression levels were higher in CMS4 samples (p<2.2e-16), consistent with previous literature showing that cancer stem cells of tumors that have undergone mesenchymal transition predominantly express the standard CD44 isoform that contains no variant exons. Finally, variance of the transcription profiles suggested potential intra-subtype heterogeneity and an opportunity to further refine these global patterns. Discussion: Here we present an in-depth transcriptome analysis of TCGA CRC data across the molecular subtypes of the disease. We were able to identify differential transcription patterns in an unbiased way by using a scalable and efficient computational approach that models both annotated and novel splice variants simultaneously. Our pipeline revealed a number of promising candidates that may be acting as key modulators in specific disease subsets and deserve further investigation. Citation Format: Yin Hu, Rodrigo Dienstmann, Justin Guinney. RNA-seq differential transcription analysis of TCGA colorectal cancer (CRC) transcriptomes reveals subtype-specific isoform usage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2015-1932

K-Ras 4A Transcript variant is up-regulated in eutopic endometrium of endometriosis patients during proliferative phase of menstrual cycle.

K-Ras transcripts comprise two main isoforms: K-Ras 4A and K-Ras 4B, which act differently. The expression of both isoforms was reported in many human tissues. However, K-Ras 4B was the major expressed transcript variant. An increased expression of K-Ras 4B mRNA was reported in eutopic endometrium of endometriosis patients. In this way, we aimed to study the expression of K-Ras 4A transcript in eutopic endometrium related to endometriosis. Employing exon4-flanking primers, K-Ras isoforms were simultaneously amplified in a RT-PCR reaction. Quantitative real-time PCR was performed using GAPDH as an internal control. K-Ras 4A transcript expression in eutopic endometrium was analyzed by ΔΔC T method. We identified existence of both of K-Ras 4A and K-Ras 4B in eutopic endometrium of patients and controls. Quantitative real-time analysis demonstrated that K-Ras 4A expression was 2.7-fold higher in endometriosis than non-endometriosis eutopic samples. Interestingly, this overexpression mainly occurs through the proliferative phase of menstrual cycle. The findings bring to light the eminent role of K-Ras 4A in endometriosis. This splice variant which is known for promoting apoptosis could be an effective factor in balance between proliferation and death of eutopic endometrial cells.

Abstract IA15: Membrane targeting of Ras

Abstract Ras proteins are targeted to membranes by virtue of post-translational modifications of their C-terminal hypervariable regions (HVRs). Because Ras proteins signal only when associated with cellular membranes, the Ras trafficking pathway is considered an attractive area for anti-cancer drug discovery. The three mammalian Ras genes, hras, nras and kras, give rise to four Ras proteins because the transcript of the kras locus is alternatively spliced to encode K-Ras4A and K-Ras4B. The kras locus is most often mutated in human cancer and when mutated gives rise to oncogenic forms of both K-Ras4A and K-Ras4B. The four Ras protein differ only in their membrane targeting HVRs. Each Ras protein ends in a CAAX sequence, which signals for a three-step modification that includes farnesylation, AAX proteolysis and carboxyl methylation. N-Ras, H-Ras and K-Ras4A are further modified by palmitoylation of one or two cysteines upstream of the CAAX sequence. Palmitoylation of N-Ras and H-Ras occurs on the cytoplasmic face of the Golgi apparatus. Whereas farensylation affords relatively weak membrane affinity, palmitoylation of the farnesylated protein provides stable association with membranes. Palmitoylation is readily reversible and a palmitoylation/depalmitoylation cycle drives a cycle of trafficking to and from the Golgi. K-Ras4B is unique in utilizing a polybasic sequence upstream of the CAAX motif to associate with the plasma membrane through an electrostatic interaction. We have shown that K-Ras4B can be phosphorylated by PKC at serine 181 in the HVR and this modification partially neutralizes the electrostatic charge such that K-Ras4B loses affinity for the plasma membrane and is relocated on endomembranes in a process we have called the farnesyl-electrostatic switch. Because phosphorylation of K-Ras4B is associated with diminished cell viability we have explored the possibility that induction of phosphorylation of oncogenic K-Ras4B may be one way to target K-Ras transformed cells. Other aspects of K-Ras biology that will be discussed are the relative expression of K-Ras4A and K-Ras4B and the unique membrane trafficking of K-Ras4A. A K-Ras4B membrane release assay suitable for siRNA and small compound screening will also be discussed. Citation Format: Frederick Tsai, Nicole Fehernbacher, Pamela Sung, Helen Court, Mark R. Philips. Membrane targeting of Ras. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA15. doi: 10.1158/1557-3125.RASONC14-IA15

Evidence for aldosterone-dependent growth of renal cell carcinoma.

The aim if this study was to investigate the hypothesis that K-RAS 4A is upregulated in a mineralocorticoid-dependent manner in renal cell carcinoma and that this supports the proliferation and survival of some renal cancers. Expression of the K-RAS in renal tumour tissues and cell lines was examined by real-time PCR and Western blot and mineralocorticoid receptor, and its gatekeeper enzyme 11β-hydroxysteroid dehydrogenase-2 was examined by immunocytochemistry on a tissue microarray of 27 cases of renal cell carcinoma. Renal cancer cells lines 04A018 (RCC4 plus VHL) and 04A019 (RCC4 plus vector alone) were examined for the expression of K-RAS4A and for the effect on K-RAS expression of spironolactone blockade of the mineralocorticoid receptor. K-RAS4A was suppressed by siRNA, and the effect on cell survival, proliferation and activation of the Akt and Raf signalling pathways was investigated in vitro. K-RAS4A was expressed in RCC tissue and in the renal cancer cell lines but K-RAS was downregulated by spironolactone and upregulated by aldosterone. Spironolactone treatment and K-RAS suppression both led to a reduction in cell number in vitro. Both Akt and Raf pathways showed activation which was dependent on K-RAS expression. K-RAS expression in renal cell carcinoma is at least partially induced by aldosterone. Aldosterone supports the survival and proliferation of RCC cells by upregulation of K-RAS acting through the Akt and Raf pathways.

Mouse pulmonary adenoma susceptibility 1 locus is an expression QTL modulating Kras-4A.

Pulmonary adenoma susceptibility 1 (Pas1) is the major locus responsible for lung tumor susceptibility in mice; among the six genes mapping in this locus, Kras is considered the best candidate for Pas1 function although how it determines tumor susceptibility remains unknown. In an (A/J×C57BL/6)F4 intercross population treated with urethane to induce lung tumors, Pas1 not only modulated tumor susceptibility (LOD score = 48, 69% of phenotypic variance explained) but also acted, in lung tumor tissue, as an expression quantitative trait locus (QTL) for Kras-4A, one of two alternatively spliced Kras transcripts, but not Kras-4B. Additionally, Kras-4A showed differential allelic expression in lung tumor tissue of (A/J×C57BL/6)F4 heterozygous mice, with significantly higher expression from the A/J-derived allele; these results suggest that cis-acting elements control Kras-4A expression. In normal lung tissue from untreated mice of the same cross, Kras-4A levels were also highly linked to the Pas1 locus (LOD score = 23.2, 62% of phenotypic variance explained) and preferentially generated from the A/J-derived allele, indicating that Pas1 is an expression QTL in normal lung tissue as well. Overall, the present findings shed new light on the genetic mechanism by which Pas1 modulates the susceptibility to lung tumorigenesis, through the fine control of Kras isoform levels.

Abstract 4116: Relationship between K-ras 4A and superoxide in lung adenocarcinoma cell lines

Abstract The K-ras gene is mutated in 30-50% of human lung adenocarcinomas, and even more frequently in cancers of colon and pancreas. It exists in two protein isoforms, K-ras 4A and 4B. Their amino acid sequence is very similar, except for marked differences in the hypervariable domain near the carboxyl terminus. K-ras 4B, with a polylysine tail, is unique among the ras gene products and is the dominant K-ras isoform in most tissues. Wild-type K-ras 4B has tumor suppressor activity (Zhang et al. Nature Gen. 29:25, 2001), whereas K-ras 4A was recently found to be necessary for lung tumor development in mice (To et al. Nature Gen. 40:1240, 2008). In search of an oncogenic function for K-ras 4A in lung cancer, we previously demonstrated a highly significant (P<0.01) positive correlation between K-ras 4A expression, at both mRNA and protein levels, and superoxide amounts in a panel of 11 human lung adenocarcinoma cell lines (Proc. Am. Assoc. for Cancer Research 50: (#437)). Superoxide has been widely implicated in cancer causation and progression. In the present study, we test whether there is a cause-effect relationship between expression of K-ras 4A and superoxide production. Cell line H441 expresses high levels of both superoxide and K-ras 4A. Superoxide levels were determined using a nitroblue tetrazolium (NBT) assay and K-ras 4A protein levels quantified by immunoblot with a specific antibody. Results are means ± SEMs. The effect of reduced levels of K-ras 4A on superoxide production was determined using a K-ras 4A silencing siRNA, which reduced K-ras 4A protein by 60.2 ± 3.0% after 72 hours of siRNA treatment. This reduction had no effect on superoxide (control 1.13 ± 0.08, siRNA 1.17 ± 0.08 nmol NBT reduced/ug protein). The effect of reduced levels of superoxide on K-ras 4A expression was determined using 5 mM Tiron, an electron scavenger. A 36% decrease in superoxide did not affect K-ras 4A protein levels (control 26.90 ± 0.89 vs. Tiron-treated 26.89 ± 1.91, arbitrary units). If these preliminary results are confirmed with additional cell lines and reagents, they may suggest that both expression of K-ras 4A and production of superoxide are controlled by another, pro-oncogenic factor or set of conditions. In view of the importance of both K-ras and superoxide in cancer, discovery of this putative central pro-oncogenic regulator might offer an important preventive and therapeutic target in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4116.

K‐ras exon 4A has a tumour suppressor effect on carcinogen‐induced murine colonic adenoma formation

K-ras encodes two isoforms, K-ras 4A and 4B, that are jointly affected by K-ras activating mutations, which are prevalent in colorectal cancer (CRC). CRC shows alterations in the expressed K-ras 4A : 4B isoform ratio in favour of K-ras 4B, in tumours both with and without K-ras mutations. The present study evaluated whether K-ras 4A expression can suppress colonic adenoma development in the absence of its oncogenic allele. Mice with homozygous targeted deletions of K-ras exon 4A (K-ras(tmDelta4A/tmDelta4A)) that can express the K-ras 4B isoform only, along with heterozygous K-ras(tmDelta4A/+) and wild-type mice, were given ten weekly 1,2-dimethylhydrazine (DMH) treatments to induce colonic adenomas. There was a significant increase in both the number and the size of colonic adenomas in DMH-treated K-ras(tmDelta4A/tmDelta4A) mice, with reduced survival, compared with heterozygous and wild-type mice. No K-ras mutations were found in any of the 30 tumours tested from the three groups. Lack of expression of K-ras 4A transcripts was confirmed, whereas the relative expression levels of K-ras 4B transcripts were significantly increased in the adenomas of K-ras(tmDelta4A/tmDelta4A) mice compared with K-ras(tmDelta4A/+) and wild-type mice. Immunohistochemical studies showed that adenomas of K-ras(tmDelta4A/tmDelta4A) mice had significantly increased cell proliferation and significantly decreased apoptosis with evidence of activation of MapKinase and Akt pathways, with increased phospho-Erk1/2 and both phospho-Akt-Thr308 and phospho-Akt-Ser473 immunostaining, compared with adenomas from K-ras(tmDelta4A/+) and wild-type mice. In conclusion, following DMH treatment, K-ras exon 4A deletion promoted increased number and size of colonic adenomas showing increased K-ras 4B expression, increased proliferation, decreased apoptosis, and activation of MapKinase and Akt pathways, in the absence of K-ras mutations. Therefore, K-ras 4A expression had a tumour suppressor effect on carcinogen-induced murine colonic adenoma formation, explaining the selective advantage of the altered K-ras 4A : 4B isoform ratio found in human colorectal cancer.

Prognostic significance of alterations in KRAS isoforms KRAS‐4A/4B and KRAS mutations in colorectal carcinoma

Somatic KRAS mutation is an early well-known event in colorectal carcinogenesis but a complete understanding of RAS function and dysfunction in colorectal cancer is still to come. Our aim was to study the incidence of KRAS mutation; KRAS splice variants: KRAS4A and KRAS4B; and their relationships with various clinico-pathological characteristics in colorectal cancer (CRC).In this study, 285 CRC cases were analysed for KRAS mutation by direct DNA sequencing followed by immunohistochemical analysis after validation with real-time PCR assay, to study the protein expression of KRAS4A and -4B isoforms. KRAS gene mutations were seen in 80/285 CRCs (28.1%) and of the mutated cases, the majority of the mutations were seen in codon 12 (81.2%) as opposed to codon 13 (18.8%). CRCs with KRAS mutations were associated with a poor overall survival (p = 0.0009). Furthermore, KRAS mutations at codon 12 were associated with a poor overall survival of 64.4% at 5 years compared with a 5-year overall survival of 75.8% and 78.2% with codon 13 mutation and absence of KRAS mutations, respectively (p = 0.0025). KRAS4A protein expression was predominantly seen in the cytoplasm, while KRAS4B protein was nuclear. KRAS4A overexpression was significantly associated with left colon, histology subtype of adenocarcinoma, p27kip1, and cleaved caspase3 expression. Interestingly, KRAS4A overexpression was associated with a better overall survival (p = 0.0053). On the other hand, KRAS4B overexpression (33.2%) was significantly associated with larger tumour size (p = 0.0234) and inversely correlated with p27kip1 protein (p = 0.0159). Both KRAS mutation and KRAS4A were independent prognostic markers in a multivariate analysis with age, gender, stage, differentiation, and MSI status. Our results highlight the differential role of KRAS isoforms in CRC, their utility as a prognostic biomarker, and underline the importance of KRAS alterations as a potential therapeutic target for CRC.

Kras regulatory elements and exon 4A determine mutation specificity in lung cancer

Kras is the most frequently mutated ras family member in lung carcinomas, whereas Hras mutations are common in tumors from stratified epithelia such as the skin. Using a Hras knock-in mouse model, we demonstrate that specificity for Kras mutations in lung and Hras mutations in skin tumors is determined by local regulatory elements in the target ras genes. Although the Kras 4A isoform is dispensable for mouse development, it is the most important isoform for lung carcinogenesis in vivo and for the inhibitory effect of wild-type (WT) Kras on the mutant allele. Kras 4A expression is detected in a subpopulation of normal lung epithelial cells, but at very low levels in lung tumors, suggesting that it may not be required for tumor progression. The two Kras isoforms undergo different post-translational modifications; therefore, these findings can have implications for the design of therapeutic strategies for inhibiting oncogenic Kras activity in human cancers.

ALTERNATIVE SPLICING OF THE K-RAS GENE IN MOUSE TISSUES AND CELL LINES

The mRNA from the K-ras protooncogene is alternatively spliced into 2 transcripts, K-ras4A and K-ras4B, which possess 2 alternative fourth coding exons. In the present study, expression of K-ras4A and K-ras4B transcripts in 8 organs, including heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis, from BALB/c mice were determined by Northern blot hybridization. K-ras4B, observed in all organs, accounted for approximately 90% to 99% of total K-ras mRNA. K-ras4A was detected only in lung, liver, and kidney. In addition, K-ras expression in lungs and K-ras4A/K-ras4B ratios in lung, liver, spleen, and kidney from A/J, BALB/c, C3H/HeJ, and C57BL/6Jmice were determined. A/Jlungs expressed K-ras mRNA 2-fold higher than C3H/HeJ or C57BL/6Jlungs, whereas K-ras mRNA expression in BALB/c lungs was intermediate. Higher percentages of K-ras4A mRNA were found in lungs and kidneys from A/Jand BALB/c mice, as compared with those from C3H/HeJand C57BL/6Jmice. Levels of K-ras4A and K-ras4B mRNAs were also examined in 20 NIH 3T3 cell lines transformed by DNA from spontaneous A/Jmouse lung tumors. K- ras4A was expressed 2-to 3-fold higher in these cell lines than in nontransformed NIH 3T3 cells and in C10 cell lines. These results suggest that: (1) there may be functional differences between the protein encoded by K-ras4A and that encoded by K-ras4B in each tissue type, and in tumor cells; and (2)K- ras mRNA expression and K-ras4A/K-ras4B ratios detected in lung tissues from different strains of mice correlate with susceptibility to tumor induction.