Dr. Angela Poff – Exploiting Cancer Metabolism with Ketosis and Hyperbaric Oxygen

Dr. Angela Poff – Exploiting Cancer Metabolism with Ketosis and Hyperbaric Oxygen

November 1, 2019 7 By William Morgan


– Thank you to the organizers
for putting this on,
I know how much work comes
behind a conference like this,
and it’s really an outstanding event
and I know everyone’s getting
a lot of value out of it.
So I am an inventor
on some USF patents in
intellectual property,
I am a scientific consultant
for an exogenous ketone company,
and I’m not a physician.
Out lab at the University of South Florida
is really interested in
developing and testing
non-toxic metabolic targeted therapies
for a variety of disorders,
but I’m most interested in
looking at those for cancer.
As you’ve heard this morning
from some of the other
speaker presentations,
many cancers seem to really
thrive on having excess glucose
and glucose signaling,
but they seem to be
vulnerable to energy stress.
So we know that there’s some evidence
that shows that high glycemic index diets
can increase the risk
of developing cancer,
that, when you have
hyperglycemia during treatment,
you can have a poor clinical outcome,
and in animal models there’s
actually been some data
to show direct correlation
between tumor growth and blood glucose
which has led other
groups, as well as our own,
to look at the ketogenic
diet as a potential way
to basically lessen this
glycolytic type of tumors.
So a few years ago we did some work
in the VMM3 mouse model
of metastatic cancers,
a very aggressive model
that metastasizes quite
rapidly throughout the body,
and we looked at two
different ketogenic diets,
both of which were able
to slow tumor growth
and prolong survival by
about 50% in these animals.
We did find that the
survival of these animals
was correlated to the ability
to decrease tumor growth
in them, but also,
our lab is extremely interested
in the role of ketones specifically,
so we wanted to take a closer look.
We knew that, from the literature,
there was data suggesting
that ketones themselves
may have direct anti-cancer effects.
We showed it in the VMM3 cells in culture
when you add exogenous ketones
onto various levels of glucose,
including even quite
high levels of glucose
such as 25 millimolar,
which is not atypical
for cell culture preparations,
you can get a reduction in the
proliferation of these cells.
This has been reported
in papers back to 1979
across a variety of
cancer cell lines as well,
and when we look at ketones themselves,
we know that there are
some characteristics
which may explain why they could be useful
as an anti-cancer agent.
So we know they stimulate
oxidative metabolism,
many tumors really thrive
on a glycolytic phenotype.
We know they inhibit inflammation, in part
through inhibiting assembly
of the NLRP3 inflammasome,
they act as HDAC inhibitors,
which we heard about yesterday morning.
HDAC inhibitors
is actually an entire
class of anti cancer drugs.
So here we have an endogenously
produced metabolite
that may be eliciting similar effects.
So they can alter the glycolytic pathways
and even directly reduce glucose.
Dr. D’Agostino mentioned this
at the end of his talk yesterday.
When we provide exogenous ketones,
we see a reduction in
blood glucose over time
that really mirrors the
elevation in ketones.
So we looked next in the same model
at a potent form of exogenous ketone,
which is a ketone ester,
and we saw that, indeed,
in this model, at least,
we did see actually
a pretty similar reduction in tumor growth
and prolongation of
survival in these mice.
These mice did exhibit some
calorie restriction effect,
so we included a calorie
restriction control group
and found that, while they
had a trend towards a benefit,
it couldn’t explain the entire benefit
that we were seeing with the ketone ester.
So in this model at least,
the ketone ester provided on
top of a standard rodent chow
was as effective as the ketogenic diet.
So, as I mentioned,
we are interested in non-toxic therapies,
and I think Dr. Chan
made a really good point
that these therapies
often seem to work best
when they’re combined.
So one thing that we wanted to evaluate
was hyperbaric oxygen.
So, when tumors grow,
they grow under very chaotic conditions,
and the blood vessels that
actually form within the tumor
are very inadequate, so
you see pockets of hypoxia
that form throughout the tumor.
This hypoxia actually
stimulates a hypoxic response
and dramatically promotes
the aggressive phenotype
of the tumor, it promotes
invasion and metastases,
reprograms metabolism,
and confers chemo radiation resistance.
So it’s thought that restoring
oxygen levels to the tumor
could be a way to re sensitize tumors
to therapies that work
via oxidative stress.
And just a quick note on
oxidative stress, cancer cells
exist in a state where
they have elevated levels
of free radicals, and that
actually benefits them
pretty greatly.
It allows them to increase mutagenesis,
and they have an adaptive
response in that way.
These free radicals can actually
also oxidize receptors for
growth factor signaling pathways
and promote growth factor signaling.
But, because they live
at this elevated state,
they’re actually closer to a threshold
where they can be damaged
by oxidative stress,
and so that’s one reason
many therapies tend to try to treat cancer
via an oxidative stress
mediated mechanism.
So hyperbaric oxygen is
delivery of 100% oxygen
at elevated pressure,
and this does increase the
amount of oxygen in the tumor.
So we then decided,
let’s combine these two
non-toxic therapies,
the ketogenic diet as
well as exogenous ketones
in the form the ketone ester
with hyperbaric oxygen
and see what we can find,
and we found a very dramatic
slowing of tumor growth,
slowing of metastatic spread,
and the animals lived twice
as long as control animals.
That was a couple of years ago,
so what I really wanted to do
is update you on what our
lab’s been doing since then.
As I mentioned, we really think
that ketosis has the potential
to sensitize tumors to other therapies,
and we want to look at
other metabolic therapies
as mono therapies, but
also in combination.
We have a number of projects going on,
I just wanted to give you a brief overview
of those different projects.
First, we have some interest
in trying to modulate tumor
metabolism with glycemic agents.
This is a project of Dr. Nathan Ward,
he was a recent PhD graduate from our lab,
he’s now a postdoc at
Moffitt Cancer Center.
Brain cancer cells in particular
really do have a robust
glycolytic metabolism,
and this makes them
quite resistant to
oxidative stress in general,
but sensitive to energy manipulation.
Two glycemic agents that
we looked at in these cells
were dichloroacetate,
which inhibits pyruvate
dehydrogenase kinase.
PDK is the inhibitor of
pyruvate dehydrogenase complex.
So basically what happens is,
when you inhibit the inhibitor of PDH,
you stimulate flow of pyruvate
into the mitochondria.
In many cancer cells,
most of it, or a lot of it
is instead going towards
lactate fermentation.
So DCA will restore oxidative metabolism
into the mitochondria of these cells.
The other agent is
metformin, which, of course,
is classically known as
an antidiabetic drug,
and we know that it inhibits
hepatic gluconeogenesis,
but it also has some molecular effects
like activating AMP kinase,
which would then inhibit PI3 Kinase,
AKT mTOR signaling,
which are all very active in most tumors
to promote protein
synthesis and tumor growth,
and it also acts as a
complex one inhibitor
of the electron mitochondrial
transport chain,
and this can induce an energetic crisis
and also induce oxidative stress.
So first in these brain cancer
derived cells, we found that
DCA does in fact activate
pyruvate dehydrogenase complex,
and we can see that it causes the cells
to produce the amount of
lactate that they’re producing,
so it’s doing what we want
it to do, which is good.
And in a dose dependent fashion
with increasing levels of DCA,
we can reduce proliferation of these cells
as well as reduce viability.
We also showed that these cells
and its effect seems to be
oxidative stress mediated
because the effect can be
rescued within acetylcysteine,
which is a cysteine donor
to help reduce glutathione.
With metformin,
we found that metformin was
cytostatic to the VMM3 cells,
meaning that it can slow
or cease proliferation
but did not effect viability.
When we combine these
two agents we found that,
even though metformin didn’t
effect cytotoxicity on its own,
we can enhance the cytotoxicity
of DCA by adding metformin,
and this effect seems to be
due to metformin’s ability
to inhibit complex one of
the electron transport chain
and generate oxidative stress.
We know this because
metformin in these cells
did not activate AMP kinase.
I’m not showing this data here,
but we could actually mimic
the same effects of metformin
using rotenone, this is
a complex one inhibitor,
and we can’t mimic the
effects using I-CAR,
which is an AMP kinase activator.
So again suggesting
that this effect that we’re
seeing in this cell line
is probably mediated by
complex one inhibition
and not AMP Kinase activation.
And this effect can be partially rescued
by an acetylcysteine
antioxidant, again suggesting
oxidative stress is probably
a contributing mechanism.
When we looked at each of
these therapies individually,
DCA and metformin at
100 migs per kig dosing
was able to pretty
dramatically slow tumor growth
in these animals, and each
increase survival time.
When we try to combine
the therapies though,
we didn’t see any additive benefit
of combining DCA with metformin,
so it’s possible that there
are redundant mechanisms
at play, or maybe in vivo we’re seeing
some kind of compensatory effect
which causes us to see the same synergy
that we found in vitro.
Another study is looking
at pharmacologic levels
of ascorbic acid, or vitamin
C, in the same model.
So this study is the
work of Jeanine Deblosi,
a very talented undergraduate student
who just graduated from USF,
and we’re losing her soon.
She’s doing a master’s at Oxford
so I can’t really blame her,
but we will miss her greatly.
Oral ascorbate can only
provide micromolar elevations
in the blood of ascorbate,
but when you deliver it by IV
you can actually get quite high levels
or pharmacological levels
up to 20 millimolar.
We all know of ascorbate’s
antioxidant effects
at these low levels,
but when you get to these very
high pharmacological levels
you can actually elicit a very
potent pro oxidative effect
in the tumor, and it doesn’t
seem to have the same effect
in healthy tissues, and for that reason
this is under investigation
in many different cancers.
There’s a few different
potential mechanisms of action
at play here.
For one, we know that ascorbate
can undergo pH dependent auto oxidation.
In the tumor microenvironment,
which is very acidic,
ascorbate will auto oxidize,
which generates hydrogen peroxide.
This can stimulate oxidative stress,
which may be useful to
damage the cancer cell.
That oxidized ascorbate
can also be taken up
by the glucose transporters
into the cancer cell.
This has a very similar
structure to glucose.
Once once inside, though,
that oxidized ascorbate
is often reduced by glutathione
and it results in an intracellular
depletion of glutathione,
which could cause these cells
to now be quite sensitize
to pro oxidative stimuli.
And, ascorbate also
is known to activate HIF hydroxylases.
These are responsible
for that hypoxic response
that causes hypoxia to
really dramatically increase
the aggressive phenotype of cancers.
So in vitro we found that ascorbate
with increasing concentrations
you get decreasing viability
and proliferation of these cells.
So again, in a dose dependent fashion.
The effect on these cells
seems to be oxidative stress mediated,
which is consistent with the literature,
again because N-acetylcysteine
can attenuate this decrease in viability.
And we can mimic the
effects that we’re seeing
with ascorbate by simply
supplying hydrogen peroxide,
so both suggesting this
is probably occurring
by oxidative stress.
So we wanted to know,
can we enhance ascorbate
induced cytotoxicity
with hyperbaric oxygen.
So we’re all about
trying to combine these
non-toxic therapies
to get a more robust
response, and the answer is,
maybe in some cancer
cells but not in others.
So we found that hyperbaric oxygen
was able to enhance the effects
in our VMM3 cell line,
which is a brain cancer cell line,
but not in U87,
which is another brain cancer cell line.
And we actually think we
may know what’s going on.
It’s possible that this difference
in the response is due to
a difference in transferrin
receptor up regulation.
So ascorbate will increase
transferrin receptor
in the VMM3 cells, but not the U87 cells.
Transferrin receptor
will cause an uptake
of iron into the cell,
which can stimulate
Fenton redox biochemistry,
and this Fenton reaction
will cause a generation
of hydroxyl radical
and other very damaging ROS molecules,
so having an up regulation in transferrin
in response to this treatment
could make those cells more susceptible
to a large pro oxidative stimulus
compared to the U87 cells,
which didn’t see a compensatory increase
in transferrin receptor.
The next study that we’re looking at
is something called press
pulse metabolic therapy.
This is an interesting idea.
Basically, tumors can,
they behave in many ways
that are quite similar to
ecological populations,
and it’s led researchers to ask,
can we apply some of the
ecological principles
of population extinction to
find new ways to target cancer?
So press disturbances,
these are chronic environmental stressors,
such as maybe famine.
Pulse disturbances would be acute events
that cause high morbidity,
like maybe a pathogenic insult.
In ecological populations,
when you have simultaneous
press and pulse disturbances,
that’s when you see
mass extinction events.
So we think that ketosis
may be a press disturbant for cancer.
It’s putting a lot of pressure
on the energy pathways
that really seem to promote robust growth
and resistance in the tumor,
and having a state of ketosis
can really reduce many of those benefits
that the Warburg effect
bestows onto the tumor.
We think this could actually be one reason
why we saw this synergy between ketosis
and hyperbaric oxygen.
We can view ketosis as
a press disturbance,
and hyperbaric oxygen, potentially,
as a pulse disturbance.
And so we want to look into this further,
because we think it could really inform
better ways of targeting
the tumor from all sides.
And, hopefully, if we’re looking
with non-toxic therapies,
we could really improve the outcome
without causing excess side effects.
So some of the press
disturbances we’re interested in
are the ketogenic diet, ketone ester,
2-deoxyglucose targets
glycolytic metabolism,
metformin, and then
some pulse disturbances
such as hyperbaric oxygen,
high dose ascorbate,
DCA, phenylacetate,
this targets glutamine metabolism,
as well as chemotherapy and radiation
would be massive pulse disturbances.
So this is very very preliminary.
This study is being led up by Sarah Moss,
who’s a really great undergraduate
researcher in our lab,
and literally the first round of this
just finished up last week
so I was analyzing the data on the plane,
but what we found so far is
we’ve tried many of these things
independently or in small combinations
in this MMTV mammary
cancer model, and so far,
the only thing that’s sticking
out is the ketogenic diet
did increase survival
time by a small amount.
But we’re excited to see, when
we combine these therapies,
are we going to have a
synergistic effect because, again,
we very typically see that
these therapies work best
in combination.
And finally, the last project
I just wanted to give you a glimpse into
is looking at modeling
and mitigating cancer cachexia.
So this is the work of Andrew Koutnik,
who is a senior PhD student in the lab,
really fantastic researcher,
and he’s at the conference,
you maybe seen him around,
and you should go up and
talk to him about his work.
He had a poster last night as well.
So cancer cachexia is a
multi-factorial syndrome
defined by an ongoing loss
of skeletal muscle mass,
with or without fat mass
that cannot be fully reversed
by conventional nutritional support
and leads the progressive
functional impairment.
So this is a big problem
in advance cancer patients.
You have large amounts of weight loss
present in up to 80% of these patients.
This weight loss really
adversely affects the
patient’s ability to receive,
tolerate, and respond to therapies,
and it’s estimated that
about 20% of cancer patients
will actually die from cachexia itself,
and there is no standard of care.
So the first question that
Andrew wanted to ask was,
does VMM3 model exhibit cachexia?
So there’s a lot of
model systems out there,
but none of them really
reproduce the entire syndrome.
And our model has a lot of benefits.
It’s syngeneic with the post background,
which means we can use
immunocompetent animals.
It’s metastatic from a primary tumor.
We can look at, we can track
it with luciferase imaging
and it’s very logistically feasible.
So we set out to evaluate
some markers of cachexia,
including a metastatic phenotype,
fat and muscle wasting,
anorexia which is present
in end stage cachexia,
inflammation, and a few other biomarkers.
I’ll have to go fast, I
don’t have a lot of time.
Indeed, this model of course
does exhibit a very severe
metastatic phenotype.
By end of life, these animals
do have reduced food intake
or anorexia, which is
consistent with cachexia.
Over time, you see that lean muscle mass
decreases over time in these animals,
so here’s gastronemius and soleus weight
and quadriceps weight decreasing,
and by the end of life
you have a very dramatic
wasting of fat mass as well.
We see increases in inflammation.
Many of the same markers
that are known to be occurring
in human cachexia patients
such as TNF alpha, IL6, splenomagaly
again suggesting an inflammatory response,
and some clinical biomarkers
like blood urea nitrogen
going up, albumin, and
total protein decreasing.
So we can say, yes, this
model seems to be actually
a very good model of cachexia.
The next step, which we have not done yet,
is to see if ketosis can
work as a mitigation strategy
in this model.
So we know that ketosis is
associated with anti inflammatory
and anti catabolic effects.
There’s even some emerging evidence
that it could be anabolic
in certain conditions.
It activated the HCA2 receptor,
which will decrease an
NF-kappa B signaling
which is known to contribute
to proteolysis in cachexia,
and we’re gonna try that ketone ester
that elevates both of the primary ketones,
VH being acetyl acetate.
So in summary, non-toxic
metabolic targeting agents
are really a promising
class of therapeutics.
Their effects are going
to be cell, cancer,
and model type dependent.
We really need to be looking at them
in combination with other
metabolic therapies,
as well as standard care,
and there is significant
metabolic heterogeneity
within tumors, and it really requires
more nuanced conversations
about efficacy of any
individual treatment.
We think that ketosis
could provide the background
for a variety of approaches
that together could put
extreme pressure on the tumor
while reducing the side
effects on the host.
So with that, thank you.
(applause)