Genetic Markers in Oncology Research Andrew Winegarner Pamplona

Genetic Markers in Oncology Research Andrew Winegarner

Pamplona, Spain

History Pamplona is the capital of the former Navarre Kingdom. A kingdom which consisted of Basque country. Which is a region of Northern Spain and Southern France with a very distinct and old language (Basque) used in addition to Spanish. Many from Basque Country have wanted independence from Spain and France for a long time.

Legacy of Hemingway visited Pamplona many times, and used the city as a setting for many of his short stories and novels. He was known for an intense interest in bullfighting. His writings inadvertently threw San Fermin into the global spotlight.

San Fermin Pamplona is best known for being where San Fermin takes place. Beginning on July 7, and going until July 14. The population of the sleepy town swells from under 200, 000 to well over a million. Everyone in the city wears white with red belt and scarf throughout the festival. It is where the infamous, Running with the Bulls takes place. Along with international fireworks competitions, hundreds of concerts and other festivities. There are rarely fatalities with the bull runs, but several will end up needing medical attention.

Universidad de Navarra The Universidad de Navarra is a private University in Pamplona, founded and operated by the Catholic organization, Opus Dei. It is known for it’s STEM degrees and its business school, which is considered the best in Spain, with satellite campuses in other cities like Barcelona. It is also a private hospital in the city. Spain has a public healthcare system, but private-care institutions like Universidad de Navarra’s Hospital are still fairly common. Generally, the quality will be nicer at private institutions, but the public facilities are still very good. Incidentally, this university has been bombed by Basque separatists several times, making it the most bombed building in Spain since the Spanish Civil War.

CIMA or the Centro de Investigacion Medica Aplicada, is a research facility attached to the Universidad de Navarra. It is across the street from the Hospital and on a hill overlooking the rest of the campus. I spent my summer working here with Dr Ruben Pio and his lab, wherein I helped characterize genetic markers for non small cell lung carcinomas. Funding for research comes from the university, government and international grants, private donors and the Opus Dei

Frizzled 1 and 7 (of the WNT pathway) My research was primarily concerned with two potential genetic markers for non-small cell lung carcinoma, the first of which was Frizzled 1 (Frizzled 7 has already been shown to play a role in adenocarcinoma) Complement factor, C 1 q, activates WNT via frizzled pathway, and has been show to be related to the aging process and we think, in several oncogenic pathways. In well-characterized adenocarcinoma cell lines A 549 and H 1299, we saw high proliferation rates with high expression of Frizzled 1 We used RNAi to knockout expression of Frizzled 1 (which binds to the 5’ end of the gene), which inhibited malignant proliferation in both cell lines. But the fzd 1 RNAi could be promiscuously binding to fzd 7, which has already demonstrated to play a role in malignant proliferation. To safeguard against the chance of promiscuous binding. I introduced a vector with the fzd 1 gene, but with no 5’ end. Creating a cell with its endogenous fzd 1 and my 5’-less fzd 1 via the vector. To do so, I had to create unique primers and several cycles of cloning and PCRs to get the desired lines. By doing so, the cell proliferation should not be deterred, even after introducing the fzd 1 RNAi, on account of the fzd 1 vector with no 5’ end. Using MTT assays, I determined proliferation to still be exponential after applying the fzd 1 RNAi, thus demonstrating that there was no promiscuous binding of the fzd 1 RNAi to fzd 7 in the initial experiments. And demonstrating a novel role of fzd 1 in adenocarcinoma proliferation. Controls were done using Scrambled and empty vectors to minimize chances of a confounding variable.

PIK 3 ca normally activates AKT which can act as an oncogene, which is normally reversed via PTEN. AKT upregulates HIFalpha and VEGF. AKT also rescues cell from apoptosis by inhibiting caspase 8 and BAD (BCL-2 death promoter) AKT also indirectly promotes m. TOR causing cell growth Additionally PIK 3 ca can cause mutations via PIP 3 which can activate CDC 42 and RACI (both of which can be reversed via PTEN) Consequently, activating mutations in PIK 3 ca can have devastating oncogenic consequences. PIK 3 ca

Inhibition of PIK 3 ca will have varying success depending on the type of mutation. The mutation I was working with was a novel one, discovered in genetic samples gathered by oncologists from around Spain. This specific PIK 3 ca mutation arose in a cancer patient after they had been receiving cancer therapy for 1 year. It has never been documented in lung cancer before, but has been rarely seen in some endometrial carcinomas. Specifically, the mutation was found at base pair #263, causing the glycine at position #88 to mutate to an alanine. If this mutation does play a role in nonsmall cell lung carcinoma, it would provide a metric for screening after initial treatment. Especially useful, as it appears to be a mutation resistant to traditional cancer treatments, and would need a specific genetictreatment to destroy. PIK 3 ca

PIK 3 ca To further characterize this mutation I first designed 2 primers to excise the mutated PIK 3 ca gene from the patient’s cells (using enzymes Nhe 1 and Xho 1). I then designed two additional primers to be inserted within the gene to help with cloning it, since the gene is massive. I then inserted the gene into my H 1436 bacteria cell line via a cloning vector, to clone novel mutation. Using the primers to determine if the vector was successfully put into the bacteria’s genome After cloning, I lysed the bacteria to get its purified genome. I then did a PCR with enzymes to cut at the two primers I put on the outside the PIK 3 ca gene, thus removing the gene from the rest of the bacteria’s genetic code. Using the internal primers, I could tag them on a gel to ensure I had the gene I was looking for. I then physically cut the gene from the gel, and purified the contents, allowing me to then put the newly cloned gene into an expression vector. I put the expression vector in a number of cell lines to determine if this particular PIK 3 ca mutation, was in fact, a novel genetic marker for cancer. We did find exceptionally high proliferations rates with this mutated gene, thus confirming our initial suspicions.

Potential Treatments My research was cut short on account of needing to return to the US for medical school, but I was able to find potential treatments for this seeming secondary mutation in cancer patients: Other labs have demonstrated specific therapies for PIK 3 ca mutations, such as mi. R-375, which directly suppresses PIK 3 ca, which has shown potential with osteosarcomas exhibiting PIK 3 ca mutations COX 2 inhibitors can activate PTEN, which can reverse several o the mechanisms of PIK 3 ca as mentioned previously. Which could be a beneficial supplementa treatment to use before radiation and other treatments.