Point Mutations Of Ras Genes Research Articles

Personalized Medicine in Medullary Thyroid Carcinoma: A Broad Review of Emerging Treatments.

Medullary thyroid carcinoma (MTC) arises from parafollicular cells in the thyroid gland, and although rare, it represents an aggressive type of thyroid cancer. MTC is recognized for its low mutational burden, with point mutations in RET or RAS genes being the most common oncogenic events. MTC can be resistant to cytotoxic chemotherapy, and multitarget kinase inhibitors (MKIs) have been considered a treatment option. They act by inhibiting the activities of specific tyrosine kinase receptors involved in tumor growth and angiogenesis. Several tyrosine kinase inhibitors are approved in the treatment of advanced MTC, including vandetanib and cabozantinib. However, due to the significant number of adverse events, debatable efficiency and resistance, there is a need for novel RET-specific TKIs. Newer RET-specific TKIs are expected to overcome previous limitations and improve patient outcomes. Herein, we aim to review MTC signaling pathways, the most recent options for treatment and the applications for personalized medicine.

RF11 | PSAT273 RET Gene Fusion In a Putative Case of Medullary Thyroid Cancer

Abstract Background Oncogenic RET gene fusions have been identified in non-small cell lung cancer and non-medullary thyroid cancers, whereas RET point mutations are the key genetic finding in both inherited and sporadic medullary thyroid cancer (MTC). The presence of a RET gene fusion provides a target for selective RET inhibitor drugs. We describe an unusual patient with suspected MTC and a positive RET gene fusion, with excellent response to targeted therapy. Clinical Case A 50-year-old male presented to our institution with cough, fatigue, heat intolerance, and flushing. His only remarkable history was stage IIIBS Hodgkin lymphoma treated with chemotherapy, in remission for approximately 6 years. CT chest revealed new bulky mediastinal and hilar adenopathy with bulky cervical nodes. VATS biopsy of a mediastinal node showed metastatic neuroendocrine neoplasm with plasmacytoid features that was immunoreactive for calcitonin, CEA and cytokeratin, but negative for TTF1 and PAX-8. The differential diagnosis included MTC or metastatic atypical carcinoid tumor. Next Generation Sequencing was negative, including RET and RAS point mutations. Subsequent testing for re-arrangements showed a KIF5B-RET gene fusion, an oncogene identified in lung adenocarcinoma. Biochemical findings showed a calcitonin level of 16,033 (normal < 10 pg/mL) and CEA 6.9 (normal < 4.7 ng/mL). Chromogranin A was 146.6 (normal < 101.16 ng/mL). ACTH and cortisol were normal. Neck US revealed a calcified 5 mm right upper pole thyroid nodule and extensive bilateral cervical nodes. A provisional diagnosis was made of MTC. Vandetanib was started. Two months later, he reported worsening orthopnea, diarrhea, and weight loss. CT chest showed marked worsening with enlarging mediastinal hilar and supraclavicular adenopathy with new moderate sized pericardial and pleural effusion. Vandetanib was discontinued and Selpercatinib started, following FDA approval. He reported prompt resolution of dyspnea and diarrhea and improved weight and energy. Calcitonin declined to <2 pg/mL, and CEA to1.6 ng/mL four months into treatment. CT neck showed marked shrinkage of lymph nodes with a persistent calcified thyroid nodule. CT chest showed marked shrinkage of mediastinal and hilar masses, with resolution of bronchial narrowing and right pleural effusion. Nine months after presentation, there were no chest symptoms and biochemical markers and imaging remained stable. Conclusion Approximately 25% of MTC tumors lack activating point mutations in RET or RAS genes. Analysis of actionable fusion mutations should be considered if there is metastatic disease. High serum calcitonin levels are not pathognomonic for MTC and tumor histology may overlap with neuroendocrine lung cancers, creating a diagnostic challenge. Depending on whether this patient's tumor originated in the thyroid gland, it may represent an unusual case of RET gene fusion in MTC. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m.

Molecular Alterations in Relation to Histopathological Characteristics in a Large Series of Pediatric Papillary Thyroid Carcinoma from a Single Institution.

Simple SummaryPapillary thyroid carcinoma (PTC) in pediatric patients shows different characteristics compared with PTC in adults. In this large mono-institutional cohort of pediatric PTCs, tumor tissue samples have been analyzed for point mutations and fusions. Overall, molecular alterations were detected in 131 out of 163 PTCs (80%). Fusion-driven PTCs showed more aggressive clinico-pathological features compared to mutation-driven and wild-type cases. These results highlight the importance of testing pediatric PTCs for gene rearrangement in routine characterization of tumors.Papillary thyroid carcinoma (PTC) presents distinct clinico-pathological and molecular differences in children compared with adult patients. Whether the presence of rearrangements or point mutations is associated with aggressive PTC clinical presentation is still controversial. In this study, PTCs diagnosed in patients aged less than 18 years were retrospectively searched from the institutional archive and tumor tissue was tested for point mutations in BRAF and RAS genes and for rearrangements in RET, NTRK1, NTRK3, ALK, PPARG, BRAF and THADA. A total of 163 PTCs were analyzed. Point mutations were found in 83 (51%) and gene fusions in 48 cases (30%). The most frequent alteration was the BRAFV600E mutation (36.8%), followed by NTRK3 fusion (11%), NRAS mutation (10.4%) and RET fusion (10.4%). Fusion-driven PTCs showed more frequently infiltrative growth, larger tumors, extrathyroidal extension and N1b disease. PTCs showing solid growth pattern were significantly enriched in gene fusions. This is one of the largest cohorts of pediatric PTCs. Fusion-driven tumors most frequently show aggressive pathological features; the search for rearrangements, especially in tumors with solid areas, could improve the characterization of pediatric PTCs and offer possible therapeutic options.

Natural Products Attenuating Biosynthesis, Processing, and Activity of Ras Oncoproteins: State of the Art and Future Perspectives

RAS genes encode signaling proteins, which, in mammalian cells, act as molecular switches regulating critical cellular processes as proliferation, growth, differentiation, survival, motility, and metabolism in response to specific stimuli. Deregulation of Ras functions has a high impact on human health: gain-of-function point mutations in RAS genes are found in some developmental disorders and thirty percent of all human cancers, including the deadliest. For this reason, the pathogenic Ras variants represent important clinical targets against which to develop novel, effective, and possibly selective pharmacological inhibitors. Natural products represent a virtually unlimited resource of structurally different compounds from which one could draw on for this purpose, given the improvements in isolation and screening of active molecules from complex sources. After a summary of Ras proteins molecular and regulatory features and Ras-dependent pathways relevant for drug development, we point out the most promising inhibitory approaches, the known druggable sites of wild-type and oncogenic Ras mutants, and describe the known natural compounds capable of attenuating Ras signaling. Finally, we highlight critical issues and perspectives for the future selection of potential Ras inhibitors from natural sources.

Molecular and IHC analysis of head and neck carcinomas associated with HPV infection.

Head and neck squamous cell carcinomas (HNSCC) are a highly heterogenous disease which can be induced by two main carcinogens - tobacco and/or alcohol, or by HR HPV infection. This work examined 60 paraffin-embedded biopsies of head and neck carcinomas after histological verification. HPV infection, including its specific types in various HNSCC areas, was studied using multiplex qPCR. Expression levels of p16INK4A and p53 were detected by subsequent IHC analysis as being potential diagnostic markers. Based on the assumption that patients with HNSCC could benefit from anti-EGFR therapy (cetuximab), but the predictors are not yet defined, analyses of point mutations of ras genes (Kras, Nras) were carried out using multiplex qPCR and sequence analysis of the Braf gene. All statistical data were processed by Chí-x2 test.HPV infection was detected in 23.34 % of cases with HNSCC, of which 100 % were HPV 16, which is the most frequently infection found in the oropharyngeal region. Using IHC analysis, a positive expression of P16INK4A was detected in 100 % of HPV-positive HNSCC while this expression was discovered to be highly correlated with HPV infection. Furthermore, a correlation between p53 and HPV-negative HNSCC was proved. The mutation incidence was the highest in the Kras gene (codon 12 and codon 146), Nras (codon 12) and Braf. A correlation between tumor location in the oropharyngeal region and Kras mutations was proved. The HPV infection correlated with Kras mutations in case of codon 146 but on the grounds of low amount of output data, these figures could be irrelevant. In one case, c.1808 G>A, protein 603 Arg>Gln mutation was found in the Braf gene but its correlation with head and neck carcinomas has not been described yet (Tab. 2, Fig. 2, Ref. 24). Keywords: head and neck carcinomas, biopsy, HPV types, PCR, p16INK4A, p53, molecular predictors, Kras, Nras, Braf.

Anaplastic lymphoma kinase (ALK) gene rearrangements in radiation‐related human papillary thyroid carcinoma after the Chernobyl accident

Childhood radiation exposure has been associated with increased papillary thyroid carcinoma (PTC) risk. The role of anaplastic lymphoma kinase (ALK) gene rearrangements in radiation‐related PTC remains unclear, but STRN‐ALK fusions have recently been detected in PTCs from radiation exposed persons after Chernobyl using targeted next‐generation sequencing and RNA‐seq. We investigated ALK and RET gene rearrangements as well as known driver point mutations in PTC tumours from 77 radiation‐exposed patients (mean age at surgery 22.4 years) and PTC tumours from 19 non‐exposed individuals after the Chernobyl accident. ALK rearrangements were detected by fluorescence in situ hybridisation (FISH) and confirmed with immunohistochemistry (IHC); point mutations in the BRAF and RAS genes were detected by DNA pyrosequencing. Among the 77 tumours from exposed persons, we identified 7 ALK rearrangements and none in the unexposed group. When combining ALK and RET rearrangements, we found 24 in the exposed (31.2%) compared to two (10.5%) in the unexposed group. Odds ratios increased significantly in a dose‐dependent manner up to 6.2 (95%CI: 1.1, 34.7; p = 0.039) at Iodine‐131 thyroid doses >500 mGy. In total, 27 cases carried point mutations of BRAF or RAS genes, yet logistic regression analysis failed to identify significant dose association. To our knowledge we are the first to describe ALK rearrangements in post‐Chernobyl PTC samples using routine methods such as FISH and IHC. Our findings further support the hypothesis that gene rearrangements, but not oncogenic driver mutations, are associated with ionising radiation‐related tumour risk. IHC may represent an effective method for ALK‐screening in PTCs with known radiation aetiology, which is of clinical value since oncogenic ALK activation might represent a valuable target for small molecule inhibitors.

A Brief Review on The Molecular Basis of Medullary Thyroid Carcinoma.

Approximately 5-10% of all thyroid cancers are medullary thyroid carcinomas (MTC). MTC is mainly sporadic in nature, but 20-30% of cases are hereditary. Genetic testing for hereditary MTC is very important for the patient and his family, but the patients must be receiving appropriate genetic counseling. About 98% of patients with hereditary MTC have germline mutations in exons 10, 11, 13, 14, 15, 16 and intron 16 of the REarrangement during transfection (RET) proto-oncogene, but the etiology of the more frequent sporadic form of MTC (sMTC) is not well understood. Recently, it has been reported that apparently sporadic MTC may involve point mutations in BRAF and RAS genes, with an overall prevalence of almost 10%. Also alteration and abnormal expression of miRNA has been described in MTC. In this review, we attempted to mention some mutations and molecular changes in sporadic and hereditary MTC pathogenesis.

Genetic Alterations in Differentiated Thyroid Cancer Patients with Acromegaly.

Acromegaly is associated with increased thyroid cancer risk. We aimed to analyze the frequency of point mutations of BRAF and RAS genes, and RET/PTC, PAX8/PPARγ gene rearrangements in patients with acromegaly having differentiated thyroid cancers (DTC) and their relation with clinical and histological features. 14 acromegalic patients (8 male, 6 female) with DTC were included. BRAF V600E and NRAS codon 61 point mutations, RET/PTC1, RET/PTC3, and PAX8/PPARγ gene rearrangements were analyzed in thyroidectomy specimens. We selected 14 non-acromegalic patients with DTC as a control group. 2 patients (14.3%) were detected to have positive BRAF V600E and 3 patients (21.4%) were detected to have NRAS codon 61 mutation. NRAS codon 61 was the most frequent genetic alteration. Patients with positive mutation had aggressive histologic features more frequently than patients without mutations. Comparison of the acromegalic and non-acromegalic patients with DTC revealed that BRAF V600E mutation was more frequent in non-acromegalic patients with DTC (14.2% vs. 64.3%, p=0.02). RET/PTC 1/ 3, PAX8/PPARγ gene rearrangements were not detected in any patient. None of the patients including the patients with positive point mutations had recurrence, and local and/or distant metastasis. NRAS codon 61 is the most frequent genetic alteration in this acromegaly series with DTC. Since acromegalic patients have lower prevalance of BRAF V600E mutation, BRAF V600E mutation may not be a causative factor in development of DTC in acromegaly. Despite the relation of BRAF V600E and NRAS codon 61 mutations with aggresive histopathologic features, their impact on tumor prognosis remains to be defined in acromegaly in further studies.

RAS proto-oncogene in medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare malignancy originating from the calcitonin-secreting parafollicular thyroid C cells. Approximately 75% of cases are sporadic. Rearranged during transfection (RET) proto-oncogene plays a crucial role in MTC development. Besides RET, other oncogenes commonly involved in the pathogenesis of human cancers have also been investigated in MTC. The family of human RAS genes includes the highly homologous HRAS, KRAS, and NRAS genes that encode three distinct proteins. Activating mutations in specific hotspots of the RAS genes are found in about 30% of all human cancers. In thyroid neoplasias, RAS gene point mutations, mainly in NRAS, are detected in benign and malignant tumors arising from the follicular epithelium. However, recent reports have also described RAS mutations in MTC, namely in HRAS and KRAS. Overall, the prevalence of RAS mutations in sporadic MTC varies between 0-43.3%, occurring usually in tumors with WT RET and rarely in those harboring a RET mutation, suggesting that activation of these proto-oncogenes represents alternative genetic events in sporadic MTC tumorigenesis. Thus, the assessment of RAS mutation status can be useful to define therapeutic strategies in RET WT MTC. MTC patients with RAS mutations have an intermediate risk for aggressive cancer, between those with RET mutations in exons 15 and 16, which are associated with the worst prognosis, and cases with other RET mutations, which have the most indolent course of the disease. Recent results from exome sequencing indicate that, besides mutations in RET, HRAS, and KRAS, no other recurrent driver mutations are present in MTC.

Abstract B36: Targeting oncogenic RAS with small molecule PKC-delta inhibitors

Abstract Somatic point mutations of RAS genes at codons 12, 13, and 61 are the most common dominant oncogenic lesions in human cancer, making aberrant RAS signaling an important therapeutic target. Unfortunately, the oncogenic RAS/GAP switch is an exceedingly difficult target for rational drug discovery. We have developed a novel, alternative strategy to circumvent this limitation of targeting RAS directly. Mutated, constitutively-activated RAS is lethal to the tumor cell unless a survival pathway, mediated by a novel PKC isozyme, PKC-delta, is also active. This ”non-oncogene” dependency of RAS-mutant cells on PKC-delta activity can be exploited therapeutically. We have demonstrated the selective susceptibility of human pancreatic, lung, colon, ovarian and neuroendocrine tumor cells with mutant K- or HRAS alleles to PKC-delta inhibition. Importantly, unlike the classical PKC isozymes, PKC-delta is not required for the survival of normal cells, and its inhibition or down-regulation in normal cells and organisms has no adverse effects. Furthermore, we demonstrate that cancer stem-like cells derived from RAS-mutant tumor specimens and cell lines are equally or more susceptible to PKC-delta inhibition as the non-stem-like population, both in culture and in xenograft studies. There is currently no treatment option that targets melanoma with NRAS mutations, which is the second most frequently mutated gene in melanoma. We report here that PKC-delta inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKC-delta were designed as chimeric hybrids of two naturally-occurring PKC-delta inhibitors, staurosporine and rottlerin. The specific hypothesis we have interrogated and validated is the concept that combining two domains of two naturally-occurring PKC-delta inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves selectivity for the specific PKC-delta isozyme. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKC-delta, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKC-delta inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKC-delta inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKC-delta inhibition effectively induced cytotoxicity in BRAF-mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKC-delta in patients who have relapsed following treatment with BRAF inhibitors. Collectively, these findings demonstrate that inhibition of PKC-delta by novel small molecule inhibitors represents a novel and targeted therapeutic approach to tumors bearing mutant RAS alleles. Citation Format: Asami Takashima, Zhihong Chen, Brandon English, Robert M. Williams, Douglas V. Faller. Targeting oncogenic RAS with small molecule PKC-delta inhibitors. [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 B36. doi: 10.1158/1557-3125.RASONC14-B36

Activation of RAS Genes by Specific Point Mutations in Transitional Cell Carcinoma of Urinary Bladder in Kashmiri Population

Urinary bladder cancer is a common malignancy in the west and ranks 7th most cancer in this region. The primary aim of the study was to evaluate the incidence of specific RAS gene point mutations among a group of Kashmiri patients diagnosed with bladder cancer. We also explored the correlation of clinic-pathological status of the illness with these mutations.The paired tumor and adjacent normal tissue specimens of 65 consecutive patients were examined in Kashmiri population,India. The DNA preparations were evaluated for the occurrence of RAS gene mutations by PCR-SCCP and DNA sequencing.Overall somatic point mutations of all the forms of RAS genes aggregated to 21.5% . In total there were nine mutations in HRAS (three in codon 12, six in codon 61), five in NRAS (two in codon 12 and three in codon 61) and no mutation was found in KRAS gene. Overall RAS genes showed no association with any clinic-pathological parameters but when stratified alone the pattern ofHRAS mutation in this study showed a significant association with smoking in bladder tumors (p <0.05).Activation of RAS gene mutation plays a less frequent role than other genetic events in the development of the most transitional cell tumors of the bladder but contribute equally well to the development of both major tumor groups. JMS 2012;15(1):15-21

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Is ionizing radiation responsible for the increasing incidence of thyroid cancer?

The dose of exposure to ionizing radiation appears to correlate with increased risk of thyroid microcarcinoma. Can ionizing radiation be a factor contributing to the increasing incidence of thyroid cancer around the world?

K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells

AbstractDefining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic KrasG12D allele on phosphorylated signaling molecules in primary c-kit+ lin−/low hematopoietic stem/progenitor cells. Comparison of wild-type and KrasG12D c-kit+ lin−/low cells shows that K-RasG12D expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas KrasG12D cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell–targeted interventions.

RAS mutations are uncommon in multiple myeloma and other monoclonal gammopathies

Monoclonal gammopathies are a group of diseases characterised by the proliferation of a single clone of plasma cells that produce a homogeneous monoclonal protein (M protein or myeloma protein) that consist of two heavy polypeptide chains of the same class and subclass and two light polypeptide chains of the same type. Multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are the most common monoclonal gammopathies. Despite advances in systemic and supportive therapies, MM is an incurable hematological malignancy with a median survival of between two and three years. Point mutations in the Ras genes can be detected in a variety of human malignancies, indicating that ras activation represents a widespread oncogenic event. Several studies have analysed the incidence of Ras mutation in MM and MGUS with great differences in their results. To date, the etiopathogenesis of these diseases is still unknown and the relevance of Ras mutation to the clinical and biological behaviour of monoclonal gammopathies remains to be elucidated. In this study, we have analysed K-ras codon 12 and N-ras codon 61 mutations on anti-CD138 sorted bone marrow plasma cell samples of 44 cases of monoclonal gammopathies: 30 MM, 13 MGUS and 1 plasma cell leukaemia, using polymerase chain reaction. No mutations within either codon 12 of K-ras or codon 61 of N-ras have been found in any of the samples. These results indicate that Ras mutations do not play a significant role in the pathogenesis of MM in the Spanish population.

Ras-mediated Loss of the Pro-apoptotic Response Protein Par-4 Is Mediated by DNA Hypermethylation through Raf-independent and Raf-dependent Signaling Cascades in Epithelial Cells

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Inhibition of Ras oncogenic activity by Ras protooncogenes.

Point mutations in ras genes have been found in a large number and wide variety of human tumors. These oncogenic Ras mutants are locked in an active GTP-bound state that leads to a constitutive and deregulated activation of Ras function. The dogma that ras oncogenes are dominant, whereby the mutation of a single allele in a cell will predispose the host cell to transformation regardless of the presence of the normal allele, is being challenged. We have seen that increasing amounts of Ras protooncogenes are able to inhibit the activity of the N-Ras oncogene in the activation of Elk in NIH 3T3 cells and in the formation of foci. We have been able to determine that the inhibitory effect is by competition between Ras protooncogenes and the N-Ras oncogene that occurs first at the effector level at the membranes, then at the processing level and lastly at the effector level in the cytosol. In addition, coexpression of the N-Ras protooncogene in thymic lymphomas induced by the N-Ras oncogene is associated with increased levels of p107, p130 and cyclin A and decreased levels of Rb. In the present report, we have shown that the N-Ras oncogene is not truly dominant over Ras protooncogenes and their competing activities might be depending on cellular context.

K- ras exon 2 point mutations in human endometrial cancer

In the present study, we screened for the K- ras exon 2 point mutations in a group of 87 gynecological neoplasms (82 endometrial carcinomas, four carcinomas of the uterine cervix and one uterine carcinosarcoma) using the non-isotopic PCR-SSCP-direct sequencing techniques. Direct sequencing analysis revealed CAA→CAC (Gln→His) K- ras codon 61 point mutations in two (2.4%) of the 82 endometrial carcinomas mentioned above. These two cases were endometrial endometrioid carcinomas at an early clinical stage of disease (stage IB and IC due to FIGO). Those endometrial carcinomas that showed K- ras exon 2 point mutations revealed a strong positivity for heterogeneous nuclear retinoblastoma protein staining; none of these, however, have had the K- ras codon 12 point mutation. In addition, there were no K- ras gene point mutations in three endometrial carcinomas lacking the Rb protein immunohistochemically. None of the cervical carcinomas tested had K- ras gene point mutations, whereas one carcinosarcoma harbored K- ras codon 61 point mutation (CAA→CAC). In conclusion, our data support the view that K- ras exon 2 point mutations are rare events in human endometrial cancer. Rb and K- ras gene abnormalities may occur independently of each other during endometrial carcinogenesis in humans.

Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC→AT mutation at the Ki-ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha-ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR-SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki-ras gene showing that the ras gene mutational pattern is species-specific. The CAA→CTA mutation, previously found at codon 61 of the Ha-ras gene in rat HCC, was observed in 5 further VC-induced HCC but was not detected in the Thorotrast-induced HCC, suggesting carcinogen-specificity. This mutation was also absent in VC-induced ASL, which supports the cell-specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF- and Thorotrast-induced tumours. Thus, a given mutation in a tumour may be carcinogen-specific but also depend on the species and the cell type. Int. J. Cancer 85:223–227, 2000. ©2000 Wiley-Liss, Inc.

Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species.

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC-->AT mutation at the Ki-ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha-ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR-SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki-ras gene showing that the ras gene mutational pattern is species-specific. The CAA-->CTA mutation, previously found at codon 61 of the Ha-ras gene in rat HCC, was observed in 5 further VC-induced HCC but was not detected in the Thorotrast-induced HCC, suggesting carcinogen-specificity. This mutation was also absent in VC-induced ASL, which supports the cell-specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF- and Thorotrast-induced tumours. Thus, a given mutation in a tumour may be carcinogen-specific but also depend on the species and the cell type.