Transcript Andrew Winegarner

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
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
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, C1q, 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 A549 and H1299, 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 fzd1 RNAi could be promiscuously binding to fzd7, 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 fzd1 gene, but with no 5’ end.
Creating a cell with its endogenous fzd1 and my 5’-less fzd1 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 fzd1 RNAi, on account of the fzd1
vector with no 5’ end.
 Using MTT assays, I determined proliferation to still be exponential after applying the fzd1 RNAi, thus demonstrating that
there was no promiscuous binding of the fzd1 RNAi to fzd7 in the initial experiments. And demonstrating a novel role of
fzd1 in adenocarcinoma proliferation.
 Controls were done using Scrambled and empty vectors to minimize chances of a confounding variable.
 PIK3ca 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
caspase8 and BAD (BCL-2 death
promoter)
 AKT also indirectly promotes
mTOR causing cell growth
 Additionally PIK3ca can cause
mutations via PIP3 which can
activate CDC42 and RACI (both
of which can be reversed via
PTEN)
 Consequently, activating
mutations in PIK3ca can have
devastating oncogenic
consequences.
PIK3ca
 Inhibition of PIK3ca 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 PIK3ca 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.
PIK3ca
PIK3ca
 To further characterize this mutation I first designed 2 primers to excise the
mutated PIK3ca gene from the patient’s cells (using enzymes Nhe1 and Xho1). 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 H1436 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 PIK3ca 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
PIK3ca 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 PIK3ca
mutations, such as miR-375, which
directly suppresses PIK3ca, which
has shown potential with
osteosarcomas exhibiting PIK3ca
mutations
 COX2 inhibitors can activate PTEN,
which can reverse several of the
mechanisms of PIK3ca as
mentioned previously. Which
could be a beneficial
supplemental treatment to use
before radiation and other
treatments.