Brownfield revitalization

Colorectal cancer (CRC) is the third leading cause of death from cancer in North America. The KRAS gene is mutated in approximately 40-50% of all CRC, and this mutation precludes treatment with promising targeted therapeutics. c-MET is a receptor tyrosine kinase that is overexpressed in ~70% of CRCs, and expression is correlated with disease progression. We hypothesized that high c-MET plus mutant KRAS would result poor survival of CRC patients, by activating unique signalling pathways that may be targeted for therapeutic purposes. To this end, we used phosphoproteomics in a KRAS mutant cell line, and identified proteins phosphorylated on tyrosine in response to HGF stimulation, including a subset of those that contain SRC family kinase consensus motifs. Small molecule inhibitors of either SRC or c-MET reduced tyrosine phosphorylation of both proteins, indicating reciprocal signalling. We chose the c-MET target p190RhoGAP for future study, as it is often ubiquitously bound to p120RasGAP via phosphorylated tyrosine. We found that RasGAP expression is mediated in part by KRAS signalling, and that expression of RasGAP could partly rescue tumourigenicity of a CRC cell line where the mutant KRAS allele has been inactivated, indicating the requirement of both mutant KRAS and RasGAP expression in this model. We then conclude by looking at CRC patient samples to determine the role of KRAS mutation in the progression and survival of CRC. We found that both KRAS and c-MET copy number are correlated to KRAS mutation status, and that c-MET polysomy plus KRAS mutation leads to worse overall survival than KRAS mutation alone. Overall, we identified novel targets of c-MET and KRAS oncogenic signaling, and identify a population which may derive the most benefit from treatments targeting both of these lesions.

Colorectal cancer (CRC) is the third leading cause of death from cancer in North America. The KRAS gene is mutated in approximately 40-50% of all CRC, and this mutation precludes treatment with promising targeted therapeutics. c-MET is a receptor tyrosine kinase that is overexpressed in ~70% of CRCs, and expression is correlated with disease progression. We hypothesized that high c-MET plus mutant KRAS would result poor survival of CRC patients, by activating unique signalling pathways that may be targeted for therapeutic purposes. To this end, we used phosphoproteomics in a KRAS mutant cell line, and identified proteins phosphorylated on tyrosine in response to HGF stimulation, including a subset of those that contain SRC family kinase consensus motifs. Small molecule inhibitors of either SRC or c-MET reduced tyrosine phosphorylation of both proteins, indicating reciprocal signalling. We chose the c-MET target p190RhoGAP for future study, as it is often ubiquitously bound to p120RasGAP via phosphorylated tyrosine. We found that RasGAP expression is mediated in part by KRAS signalling, and that expression of RasGAP could partly rescue tumourigenicity of a CRC cell line where the mutant KRAS allele has been inactivated, indicating the requirement of both mutant KRAS and RasGAP expression in this model. We then conclude by looking at CRC patient samples to determine the role of KRAS mutation in the progression and survival of CRC. We found that both KRAS and c-MET copy number are correlated to KRAS mutation status, and that c-MET polysomy plus KRAS mutation leads to worse overall survival than KRAS mutation alone. Overall, we identified novel targets of c-MET and KRAS oncogenic signaling, and identify a population which may derive the most benefit from treatments targeting both of these lesions.

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SANTRAUKA Darbo autorė: Elena Džervienė Darbo vadovas: prof. dr. Arvydas Laurinavičius Vilnius, 2011 m. Pagrindinės sąvokos: KRAS geno mutacijos, TL-PGR. Tyrimo tikslas - įvertinti dažniausias KRAS geno mutacijas tikro laiko polimerazės grandininės reakcijos metodu pacientams, sergantiems išplitusiu storosios žarnos vėžiu. Tyrimo uždaviniai: 1. Įvertinti dažniausių KRAS geno mutacijų paplitimą ir įvairovę. 2. Įvertinti dažniausių KRAS geno mutacijų ryšį su paciento lytimi ir amžiumi, klinikine diagnoze. 3. Įvertinti įdiegtų KRAS geno mutacijų nustatymo testų patikimumą. Tyrimo populiacija. Pacientai, sergantys išplitusiu storosios žarnos vėžiu visoje Lietuvoje. Tyrimo metodai. Dažniausių KRAS geno mutacijų nustatymas TL-PGR metodu, KRAS geno mutacijų nustatymas PGR ir atvirkštinės hibridizacijos metodais. Statistinė analizė atlikta naudojant „Microsoft Office Excel 2003“ ir SPSS 13,0 for Windows versijos statistinę programą. Darbo rezultatai ir išvados. Ištyrus 24 pacientus, KRAS genas buvo mutavęs 9 (37,5 proc.) atvejais, nemutavęs - 15 (62,5 proc.) atvejų. Dažniau (po 3 kartus) pasikartojo dviejų tipų mutacijos: p.Gly12Asp ir p.Gly12Cys. P.Gly12Cys mutacija dažniau pasitaikė jaunesniojo (3 kartus), p.Gly12Asp - vyresniojo amžiaus pacientų grupėje (2 kartus). KRAS geno mutacija santykinai dažniau nustatyta moterims, nei vyrams. Mutacijų dažnis abiejose amžiaus grupėse buvo panašus (p<0,05). Pacientų, sergančių tiesiosios 13 (54 proc.) žarnos vėžiu, buvo šiek tiek daugiau... [toliau žr. visą tekstą] / SUMMARY COMMONLY KRAS GENE MUTATION TESTING IN PATIENTS WITH METASTATIC COLORECTAL CANCER BY REAL TIME POLYMERASE CHAIN REACTION Master‘s degree final scientific research work Author of the Master‘s degree scientific research work: Elena Džervienė Head of the Master‘s degree scientific research work: prof. dr. Arvydas Laurinavičius Vilnius, 2011 Keywords: KRAS gene mutation, RT-PCR. The aim of the research work was to evaluate the commonly KRAS gene mutations in patients with metastatic colorectal cancer by real time polymerase chain reaction. The main goals of the work: 1. To evaluate incidence and diversity of commonly KRAS gene mutations. 2. To evaluate links among commonly KRAS gene mutations, patients sex, age and clinical diagnosis. 3. To evaluate the raliability of KRAS gene mutations tests. Survey methods. Commonly KRAS gene mutation testing by RT-PCR and reverse hybridization methodes. Statistical analysis made using MS Office Excel 2003 and SPSS 13.0 for Windows version statistical program. Results and conclusions. After survey, in 9 of 24 cases, KRAS gene were mutated, in 15 cases – wild type. Two types of mutation repeated more recently (3 times): p.Gly12Asp and p.Gly12Cys. More often (3 times) p.Gly12Cys mutation were found in younger patients group; p.Gly12Asp mutation (2 times) - in older ones. KRAS gene mutation relatively more often were set in womens, than in mens group. KRAS gene mutation rate were similar in both age groups (p<0,05). There were more... [to full text]

With the lighting speed revolution of technologies in chemistry and biology, increasing number of proteins which eluded scientists' efforts to block them before and were labeled as `undruggable', were successfully targeted with small molecule inhibitors. During the past five years, I have been working on figuring out the path to inhibit two elusive cancer targets: Her3 and KRas. / Chemistry and Chemical Biology

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