PCR bias Toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k- ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near- sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerase. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Ran is a potential therapeutic target for cancer cells with molecular changes associated with activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways

Purpose Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities. Experimental Design Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers. Results Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing-induced apoptosis. K-Ras-mutated, c-Met-amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing-induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Conclusion Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. ©2011 AACR.

Invasive and metastatic properties of MCF-7 cells and ras(H)-transfected MCF-7 cell lines

In vitro invasion and in vivo metastasis assays were performed with a panel of MCF-7 cells transfected with isogenic constructs of mutated ras(H) genes. Both increased levels of ras(H) expression and ras(H) oncogene activation increased activity of derivative cell lines in in vitro invasion assays. In vivo formation of spontaneous metastases was assessed after intradermal inoculation of MCF-7 cells in the vicinity of the mammary fat pads of ovariectomized nude mice. No metastases were seen in the absence of estradiol treatment of the mice. With estradiol supplementation of the mice both the ras(H)-transfected and control transfected cell lines gave a higher incidence of metastases than parental MCF-7 cells. Prolonged treatment of mice with exogenous estradiol (60 days vs. 21 days) resulted in more frequent metastases to liver and lung at the end of the 90-day observation period. In contrast to activated ras(H)-gene enhancement of metastatic capacity of rodent fibroblast and epithelial cell lines, there was no correlation of ras(H) expression with in vivo metastatic capacity of a human mammary carcinoma cell line.

Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.

MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

Mutations of K-ras have been found in 30-60% of colorectal carcinomas and are believed to be associated with tumor initiation, tumor progression and metastasis formation. Therefore, silencing of mutant K-ras expression has become an attractive therapeutic strategy for colorectal cancer treatment. The aim of our study was to investigate the effect of microRNA (miRNA) molecules directed against K-ras (miRNA-K-ras) on K-ras expression level and the growth of colorectal carcinoma cell line LoVo in vitro and in vivo. In addition, we evaluated electroporation as a gene delivery method for transfection of LoVo cells and tumors with plasmid DNA encoding miRNA-K-ras (pmiRNA-K-ras). Results of our study indicated that miRNAs targeting K-ras efficiently reduced K-ras expression and cell survival after in vitro electrotransfection of LoVo cells with pmiRNA-K-ras. In vivo, electroporation has proven to be a simple and efficient delivery method for local administration of pmiRNA-K-ras molecules into LoVo tumors. This therapy shows pronounced antitumor effectiveness and has no side effects. The obtained results demonstrate that electrogene therapy with miRNA-K-ras molecules can be potential therapeutic strategy for treatment of colorectal cancers harboring K-ras mutations. © 2010 Nature Publishing Group All rights reserved.

Kirsten ras mutations in patients with colorectal cancer : the ‘RASCAL II’ study

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes’ C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes’ B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Gamma tocotrienol targets tyrosine phosphatase SHP2 in mammospheres resulting in cell death through RAS/ERK pathway

Background There is increasing evidence supporting the concept of cancer stem cells (CSCs), which are responsible for the initiation, growth and metastasis of tumors. CSCs are thus considered the target for future cancer therapies. To achieve this goal, identifying potential therapeutic targets for CSCs is essential. Methods We used a natural product of vitamin E, gamma tocotrienol (gamma-T3), to treat mammospheres and spheres from colon and cervical cancers. Western blotting and real-time RT-PCR were employed to identify the gene and protein targets of gamma-T3 in mammospheres. Results We found that mammosphere growth was inhibited in a dose dependent manner, with total inhibition at high doses. Gamma-T3 also inhibited sphere growth in two other human epithelial cancers, colon and cervix. Our results suggested that both Src homology 2 domain-containing phosphatase 1 (SHP1) and 2 (SHP2) were affected by gamma-T3 which was accompanied by a decrease in K- and H-Ras gene expression and phosphorylated ERK protein levels in a dose dependent way. In contrast, expression of self-renewal genes TGF-beta and LIF, as well as ESR signal pathways were not affected by the treatment. These results suggest that gamma-T3 specifically targets SHP2 and the RAS/ERK signaling pathway. Conclusions SHP1 and SHP2 are potential therapeutic targets for breast CSCs and gamma-T3 is a promising natural drug for future breast cancer therapy.

United Arab Emirates

The United Arab Emirates (UAE) is part of the geographic region known as the Middle East. With a land mass of 82,000 square kilometres, predominantly desert and mountains it is bordered by Oman, Saudi Arabia and the Arabian Gulf. The UAE is strategically located due to its proximity to other oil rich Middle Eastern countries such as Kuwait, Iraq, Iran, and Saudi Arabia. The UAE was formed from a federation of seven emirates (Abu Dhabi, Dubai, Sharjah, Ras Al Khaimah, Ajman, Fujuriah, and Um Al Quain) in December 1971 (Ras Al Khaimah did not join the federation until 1972) (Heard-bey, 2004, 370). Abu Dhabi is the political capital, and the richest emirate; while Dubai is the commercial centre. The majority of the population of the various Emirates live along the coast line as sources of fresh water often heavily influenced the site of different settlements. Unlike some near neighbours (Iran and Iraq) the UAE has not undergone any significant political instability since it was formed in 1971. Due to this early British influences the UAE has had very strong political and economic ties with first Britain, and, more recently, the United States of America (Rugh, 2007). Until the economic production of oil in the early 1960’s the different Emirates had survived on a mixture of primary industry (dates), farming (goats and camels), pearling and subsidies from Britain (Davidson 2005, 3; Hvit, 2007, 565) Along with near neighbours Kuwait, Bahrain, Oman, Qatar and Saudi Arabia, the UAE is part of the Gulf Cooperation Council (GCC), a trading bloc. (Hellyer, 2001, 166-168).

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

MATLAB toolboxes : robotics and vision for students and teachers

In this column, Dr. Peter Corke of CSIRO, Australia, gives us a description of MATLAB Toolboxes he has developed. He has been passionately developing tools to enable students and teachers to better understand the theoretical concepts behind classical robotics and computer vision through easy and intuitive simulation and visualization. The results of this labor of love have been packaged as MATLAB Toolboxes: the Robotics Toolbox and the Vision Toolbox. –Daniela Rus, RAS Education Cochair

Active Notch1 confers a transformed phenotype to primary human melanocytes

The importance of mitogen-activated protein kinase signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf, yet clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions showed that Notch activity is significantly higher in melanomas than their nontransformed counterparts. The use of a constitutively active, truncated Notch transgene construct (N(IC)) was exploited to determine if Notch activation is a "driving" event in melanocytic transformation or instead a "passenger" event associated with melanoma progression. N(IC)-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma, such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. N(IC)-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth, suggesting that Notch alone is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene. This new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease.

A genome-based strategy uncovers frequent BRAF mutations in melanoma

Using a genome-scanning approach to search for oncogenes, a recent report identifies somatic mutations in the signaling gene BRAF that are particularly prevalent in melanoma.

High frequency of BRAF mutations in nevi

To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.

Microarray expression profiling in melanoma reveals a BRAF mutation signature

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.