Cancer-killing viruses

We learned last time that cancer cells are cells whose DNA has been damaged beyond repair. Somatic mutations have accumulated to the point that the cell regulatory mechanisms no longer function, causing uncontrolled growth and proliferation. Despite being anomalous, cancer cells are still part of what the immune system recognizes as "self", which makes finding a cure for cancer such a hurdle. Therapy, when available, is often invasive and debilitating because the only way to make sure that all cancer cells in the body are destroyed is to stop all cells, even healthy ones, from growing. Drugs targeted at the tumor tissue only are a good alternative, though they still need to be perfected. Another way to overcome the hurdles is to train our immune system to recognize cancer cells and destroy them. In the past, I've discussed ways to do this through gene therapy and cancer vaccines.

So when my friend Mike Martin sent me this story, I thought, "Nice. Another cancer vaccine success story." As I read through, though, I realized that this wasn't quite a vaccine. It was a deadly virus turned into a "good" virus.

This is the story of the "redemption" of the poliovirus. :-)

Viruses hijack cell machinery (proteins) in order to reproduce. They do so because first of all, they are very small and they can't possibly package all the proteins they need into their tiny shell. Also, by using the cell's proteins instead of viral ones they disguise themselves: less viral proteins means more chances to evade the host immune system. When successful, most viruses end up killing their host cell.

What if we could do the opposite? What if we could hijack the viral proteins, instead, and use their "killing" machinery to ... kill cancer cells? That's the brilliant idea Dr. Matthias Gromeier, from Duke University had, and the basis of his research on oncolytic viral immunotherapy.

An oncolytic virus is a virus that targets cancerous cells. The term was coined after reports of cancer remissions that coincided with a viral infection or a vaccination. While in vitro models had originally given good results, the in vivo use of oncolytic viruses has shown to be more challenging than originally anticipated due to the complicated relationship between a virus and its host. One thing that makes the immune system so fascinating and yet so complicated to study, is that it depends not only on genetics ("innate immunity", the immunity we are born with), but also on "experiences" and "exposures" ("acquired immunity," the immunity that results from exposure to pathogens and immunogens throughout our lifetime), which are often much harder to reconstruct and fold into a model. So, whenever you try to use a virus for therapy, as in viral vectors for gene therapy, for example, you face the obstacle of different immune systems, some of which may have encountered the virus (or a similar one) before and will promptly destroy it.

In a 2011 paper [1], Gromeier and his group described PVSRIPO, a prototype nonpathogenic poliovirus they designed to treat glioblastoma, one of the most common and most aggressive brain tumors. The prototype is a poliovirus recombinant engineered to replicate exclusively in malignant cells. It targets one protein in particular, Necl-5, a tumor antigen expressed by many tumor cells. Think of it as a red flag that the tumor cells carry. PVSRIPO is able to "see" the red flag and attack the cell, eliciting "efficient cell killing and secondary, host-mediated inflammatory responses directed against the infected tumor [1]." In other words, not only it kills the cell, it also elicits immune responses against the affected area.

The prototype has been FDA-approved and is currently being tested in clinical trials with patients with glioblastoma multiforme, though it already made news:

"Of the seven others who later enrolled in Dr. DesJardins' clinical trial, one patient responded like Lipscomb [whose brain tumor is shrinking and has survived cancer for a year and a half, four times longer than most people with her type of tumor]. Two patients, whose immune systems were already severely damaged, did not. It’s too early to tell with the remaining three patients, but animal studies suggest that once the body recognizes and destroys the tumor, it won’t return. If those results hold up, researchers hope to apply the same technique to a whole range of other cancers, including melanoma and prostate cancer [2]."

[1] Christian Goetz, Elena Dobrikova, Mayya Shveygert, Mikhail Dobrikov & Matthias Gromeier (2011). Oncolytic poliovirus against malignant glioma Future Virology DOI: 10.2217/fvl.11.76

Is there such thing as over-editing?

 A while ago I wrote a post based on JS Mattick's work [1] on RNA editing, the introduction of changes in RNA molecules after they have been translated from a gene. This kind of editing confers a certain adaptability to the protein without changing the gene that codes for it. Bacteria and viruses, for example, undergo extensive RNA editing in order to constantly re-adapt to the host's immune response. In eukaryotes RNA editing is rarer, but it still happens and is involved in many epigenetic mechanisms. It is also important in the immune system, as successful immune responses are driven by a great adaptability to new invaders.

RNA editing can be obtained through the insertion of one or more nucleotides, or the opposite, the deletion of one or more nucleotides. It can also be obtained by changing a single nucleotide in a certain motif, which is carried on by special enzymes. One family of such special enzymes is the APOBEC family, some of which have an important role in defending us from retroviruses, the viruses that carry RNA.

This is how APOBEC3 enzymes operate: in order to reproduce, the retrovirus transforms its RNA into single stranded DNA and then uses an enzyme to insert its DNA into the cell's DNA. Once there, the virus will reproduce using the cell's own duplication mechanisms. That's when the APOBEC enzymes get into action, by inducing a number of mutations in the viral DNA that end up deactivating it.

So, these APOBEC enzymes are the good guys, right?

Alexandrov et al. found out that they may not be, as they explain in a recent Nature paper [2].

The authors' objective was to characterize somatic mutations in cancer tissues. As you know, cancer originates from cells with anomalies in their DNA. Some anomalies are present from birth, though the vast majority accumulate as we age, some caused by external agents known to disrupt cell regulation and DNA's ability to self-repair. Other mutations appear randomly as cells undergo cellular division. As the authors say, "different mutational processes often generate different combinations of mutation types, termed signatures." Characterizing the "mutational signatures" that are associated with cancer can help us understand the mechanisms that drive cancer growth and pave the road to better ways to target and/or prevent the disease.

Here's a summary of what the researchers found:

"We compiled 4,938,362 somatic substitutions and small insertions/ deletions (indels) from the mutational catalogues of 7,042 primary cancers of 30 different classes (507 from whole genome and 6,535 from exome sequences). In all cases, normal DNA from the same individuals had been sequenced to establish the somatic origin of variants. The prevalence of somatic mutations was highly variable between and within cancer classes, ranging from about 0.001 per megabase (Mb) to more than 400 per Mb. Certain childhood cancers carried fewest mutations whereas cancers related to chronic mutagenic exposures such as lung (tobacco smoking) and malignant melanoma (exposure to ultraviolet light) exhibited the highest prevalence [2]."

In order to catalogue the somatic changes driven by cancer, the researchers harvested both healthy and cancerous cells and compared the latter to the former. The healthy DNA was used as a reference and mutations away from this references were assumed to have originated from the disease. They compiled a list of all mutations that were statistically associated to cancer and looked at the biological pathways/mechanisms these mutations affected.

The finding that childhood cancers carried less mutations is not too surprising since cellular lineages are younger. I'm also speculating that childhood cancers are more likely to be caused by underlying genetic anomalies, maybe combined by additional somatic mutations, but since they appear earlier in life, they probably require less somatic mutations to be triggered.

Alexandrov et al compiled a table of the 21 most observed signatures across the 30 different classes of cancers and then tested them for possible statistical associations. The most common signature (60% of cancers) was associated with age. Others were associated with smoking, UV light, BRCA1/2, etc. But here's what I found surprising: two of those signatures, present in 14.4% and 2.2% of cancers respectively, were associated with APOBEC.

"On the basis of similarities in mutation type and sequence context we previously proposed that signature 2 is due to over activity of members of the APOBEC family of cytidine deaminases, which convert cytidine to uracil, coupled to activity of the base excision repair and DNA replication machineries. [. . .] However, the reason for the extreme activation of this mutational process in some cancers is unknown. Because APOBEC activation constitutes part of the innate immune response to viruses and retrotransposons it may be that these mutational signatures represent collateral damage on the human genome from a response originally directed at retrotransposing DNA elements or exogenous viruses. Confirmation of this hypothesis would establish an important new mechanism for initiation of human carcinogenesis [2]."

I found this extremely intriguing. What causes the over-expression of the APOBEC enzymes in cancer tissue? We know these enzymes become activated in response to a retroviral infection, could their over-expression be the aftermath of a viral infection, then? And then their over-activation led to over-editing and hence DNA damage? Would it be at all possible that the DNA damage that led to cancer came first instead, and then the APOBEC enzymes became activated at a later stage as an attempt from the immune system to get rid of the cancerous cells?

Clearly, more studies are needed to find the answer. A complete list of mutational signatures in cancer should be compiled and compared to known models of DNA mutagens and perturbations of the cell-repair machinery. But such list should also be correlated with the biological characteristics of each cancer, the pathways and molecular mechanisms they interact with, and of course the epidemiological changes they may induce.

[1] Mattick JS (2010). RNA as the substrate for epigenome-environment interactions: RNA guidance of epigenetic processes and the expansion of RNA editing in animals underpins development, phenotypic plasticity, learning, and cognition. BioEssays : news and reviews in molecular, cellular and developmental biology, 32 (7), 548-52 PMID: 20544741

[2] Ludmil B. Alexandrov, Serena Nik-Zainal, David C. Wedge, Samuel A. J. R. Aparicio, Sam Behjati, Andrew V. Biankin, Graham R. Bignell, Niccolò Bolli, Ake Borg, Anne-Lise Børresen-Dale, Sandrine Boyault, Birgit Burkhardt, Adam P. Butler, Carl et al. (2013). Signatures of mutational processes in human cancer Nature DOI: 10.1038/nature12477

Waterscapes

I love New Mexico, the landscape out here is unique: the land is red, the skies are stark blue during the day and then blush into the most vibrant palettes of orange and purple in the evening. Yet I do miss one thing: waterscapes.

Second Beach, La Push, Washington:

I processed the photos above in black and white because, while I was hoping for a fantastic sunset, turns out, this place is most of the time wrapped in fog, hence no colors whatsoever. I should've known since, incidentally, this is the place where the Twilight saga is set. 

Punch Bowl Waterfalls, Columbia Gorge, Oregon:

Panther Creek Falls, Columbia Gorge, Washington:

Multnomah Falls, Columbia Gorge, Oregon (with a little pixie dust added):

The pixie dust is actually water spray on the lens and it was totally NOT intentional. :-)

All pics are long exposures (10-30 seconds of exposure) except for the black and white pictures (forgot the darn tripod!), which is how one achieves the velvety texture in the water. The bluish hues come from lowering the blue luminosity slider in Lightroom. 

Sadly, no luck with star gazing and meteor showers. 

In case you're in town...

... my second collective, this year. This one promises to be a lot of fun! :-)

Opening reception this Friday at the Fuller Lodge Art Center.

What have you been up to, Elena?

You mean besides creating funky images? ;-)

Right... Well, you know, the usual.

Work: finishing up the analyses for a paper, addressing the ever-discontented reviewer number three (why is it always reviewer number three?) for another paper, submitting an abstract for a meeting in the fall.

Writing: almost done with my new story, woo-hoo! Planning on shipping off to beta readers by the end of September and have it on Lovely Agent's desk by November.

Photography: getting ready for my first personal show in Santa Fe in the fall! Very excited. :-) Though I should start an inventory pretty soon, before the basement gets completely filled up by prints and frames.

So, that's basically it. What have you guys been up to? :-)

Dreams I have

"If you could make a wish, one wish only..."
"I want to travel to outer space inside a guitar."
"A guitar? Why a guitar?"
(Smiling). "Because I can."

Also, in case you never noticed, guitars float, too. :-)

On a happy note, my four submissions for the Trickster exhibit (the first four in the post) have been accepted. Yay!

Hope everybody's having a great summer. The science posts will resume next month.

Milky Way Galore!

I swapped an old lens for a fish-eye (the Sigma 15mm f2.8) so I could finally get some decent Milky Way shots. I got the lens last month, actually, but first I had to wait for the moon to wane, then when the moon started rising late enough, monsoon started (which is a beautiful thing, not complaining!) and the sky was always cloudy at night. Finally, last night it cleared enough to show a glimpse of the Milky Way...

The first two were shot at f2.8, ISO 800 and 30 seconds of exposure, the last one at 25 seconds. The lights you see in the distance are from Santa Fe, and the clouds are the remnants of a spectacular thunderstorm.

Print available here.
Check-out my upcoming thriller CHIMERAS, coming April 2014. 

Fourth of July Fireworks

Turns out, it's not so hard to take good firework shots! :-)

Our flag was lowered because of the tragic loss of 19 young firefighters in Arizona, last Saturday. I found out last night that they had been part of the thousands of heroes that had come out here two years ago to save our town from Las Conchas Fire. These shots are dedicated to them and their families. There are no words to express how saddened I am by this loss.

Playing tricks with images

Hope you're having a great summer if you're in the northern hemisphere, winter if you're in the southern one.

Last Friday was the opening reception of Wallflowers, the current exhibit at the Fuller Lodge Art Center, where I got to enjoy amazing artwork and meet other artists -- it was great fun! While I was there I also read the call for their next exhibit. The title, "Trickster," got me thinking... Magritte, Dali', Escher... so many great minds in the past played visual tricks with their images! What does the word "Trickster" make you think of?

I'm just catching up on the science, so in the meantime, I thought I'd leave you with a few images I recently created. Hope you'll enjoy them!

Peacock by EEG

Escher's Glass by EEG

Forever by EEG

Ceci n'est pas une fraise by EEG

 Thelxiepeia by EEG

Because this makes me smile every time I watch it: the inner life of a cell

I can't remember if I've already shared this video here, but if I have, it's worth seeing more than once. In fact, I watch it every time I get frustrated at work. Every so often we get caught up in failed experiments, dead calculations, politics, grants, etc., and we forget why we are doing this: because deeply inside there's the mysterious, magical beauty of what makes life possible: the cell.

"Created by XVIVO, a scientific animation company near Hartford, CT, the animation illustrates unseen molecular mechanisms and the ones they trigger, specifically how white blood cells sense and respond to their surroundings and external stimuli."

We read about all these mechanisms in textbook, but this video brings them to life, showing you the dynamics, the landscapes, the interactions. It wows me every time. You can read the full story about the video here.

The virus-antibody arms race

One of the new concepts I learned when I started working on HIV was the most recent common ancestor, or MRCA. When you look at the genetic make-up of a population, you will find a certain amount of variety but also a much greater amount of overlap, i.e. stretches of DNA that are identical throughout the population. Using phylogenetics, one can look at these patterns of shared vs. mutated stretches, and reconstruct the genetic ancestor of the population. For example, you've probably heard of Mitochondrial Eve: since we all inherit our mitochndrial DNA from our mothers, scientists have been able to look at the mitochondrial DNA across all populations and determine the one ancestor (our common mother, so to speak) from which they all originated. Pretty cool, right?

My line of work, for the past 6-7 years has been estimating most common recent ancestors, or MRCAs, of HIV-1 populations. A few years ago we found that in sexually transmitted infections only a handful of viruses are able to come across the genital mucosa and start the infection. Therefore, if you draw a blood sample early enough (a few weeks) after the start of the infection, from that sample we can infer the MRCA of the viral population in the patient. This is particularly relevant because in the case of a viral infection, the MRCA is likely to be the virus that initiated the infection. As the infection progresses, the viral population changes, but it is the ones that are able to break the mucosal barrier (i.e. the MRCAs) that a vaccine needs to target.

Once inside the host, viral evolution is (for the most part) driven by the host's immune system as it tries to counter-attack the infection. At the same time, as the virus changes its genetic make-up to escape the immune pressure, the immune system itself changes and tries to come up with new ways to neutralize the enemy. It's an arms race that in HIV infections typically sees the immune system always one step behind: the first antibodies found in an HIV-1 infected person react with the first, unmutated virus that initiated the infection (the MRCA). As the infection progresses and the virus evolves, new antibodies are made that are able to react to the following viral generations, but typically there's always a subpolulation of viruses that's one step ahead of the antibodies and can still escape. (I hope this part is clear, I've been struggling quite a bit to find the right wording for this paragraph, so if it's not clear feel free to ask questions in the comments.)

In order to design an efficient vaccine, we need to find a way to elicit broad neutralizing antibodies, where by "broad" we mean antibodies that react not only to the present or past viral generations in one host, but to a wide variety of viruses across different hosts and populations. Such antibodies are found in a minority of HIV-infected patients and, typically, by the time they arise, the infection is so spread that they cannot clear the virus.

Ideally, a vaccine should boost a "short-cut" in the evolutionary path that leads to the production of broadly neutralizing antibodies much faster than our bodies are currently capable of. Unfortunately, all vaccine trials attempted so far have not been able to elicit broad neutralizing antibodies. Why?

Antibodies are made by B-cells, white blood cells produced in the bone marrow. In order to produce antibodies, B cells need to be activated, which happens when they find an antigen specific to their receptor. Once activated, B cells not only start producing antibodies, but they also either become memory cells (so that if the antigen is encountered again, the immune system will know which antibodies to produce in order to clear it) or they undergo further differentiation. This process of undergoing more differentiations ensures that the "match" between receptor and antigen becomes tighter and tighter. It takes many cycles of differentiations to produce HIV-1 broadly neutralizing antibodies, and, currently, the process takes so long that most patients don't produce them ever, and the ones that do, don't get them in time to clear the infection.

One reason why we believe it takes many differentiations to make HIV broadly neutralizing antibodies is that they share many similarities to self-reacting antibodies, antibodies that are normally destroyed by the body because they carry a high risk to originate auto-immune disorders (when the immune system attacks its own self instead of antigens). So, instead of eliciting the actual antibodies, could a vaccine elicit its ancestor? Remember how I said that the viral population constantly evolves and, hand in hand, so do the antibodies? Since we can estimate the viral ancestors, can we do the same for the antibodies? Can we reconstruct the differentiation pathway that leads to broadly neutralizing antibodies?

In [1], Liao and colleagues have reconstructed the lineage of the infecting virus in one African HIV-infected patient (CH505), as well as the lineage of an antibody, found in the same patient, able to neutralize 55% of ~200 HIV-1 isolates. the researchers effectively reconstructed the coevolution of virus and antibody within the patient. The patient was followed from week 6 after the infection up until 236 weeks after the infection, and during this period no antiretroviral therapy was administered. This is important because it means that the viral evolution was driven solely by the immune pressure.

Liao et al. found that the first unmutated ancestor in the B-cell lineage appears at week 14 after the infection, and it keeps mutating in ways that are reflected in the evolution of the virus. Once they retraced all the intermediate steps that led to the production of the broadly neutralizing antibody, the researchers tested all of the intermediate antibodies for reactivity against the virus, from the infecting strain to its later generations. They found that breadth and strength of reactivity increased as the antibody lineage evolved. In light of what I tried to explain above, this is a fantastic step forward in understanding how the virus evolves under the immune pressure, as it can help design a vaccine that elicits antibodies that are one step ahead (instead of behind) in the virus-host arms race.

"Thus, a candidate vaccine concept could be to use the CH505 transmitted/founder Env or Env subunits (to avoid dominant Env non-neutralizing epitopes) to initially activate an appropriate naive B-cell response, followed by boosting with subsequently evolved CH505 Env variants either given in combination, to mimic the high diversity observed in vivo during affinity maturation, or in series, using vaccine immunogens specifically selected to trigger the appropriate maturation pathway by high-affinity binding to the unmutated common ancestor and antibody intermediates. [. . .] The finding that the transmitted/founder Env can be the stimulator of a potent BnAb and bind optimally to that broadly neutralizing antibody unmutated common ancestor is a crucial insight for vaccine design, and could allow the induction of broadly neutralizing antibodies by targeting unmutated common ancestors and intermediate ancestors of broadly neutralizing antibody clonal lineage trees."

Of course, there's the usual caveats: will this kind of pathway be reproducible in other patients? How much of it is randomness and how much is it not only retraceable but reproducible is something we will only understand by getting more data from more patients. But it's a start, and a very promising one.

[1] Liao, H., Lynch, R., Zhou, T., Gao, F., Alam, S., Boyd, S., Fire, A., Roskin, K., Schramm, C., Zhang, Z., Zhu, J., Shapiro, L., Becker, J., Benjamin, B., Blakesley, R., Bouffard, G., Brooks, S., Coleman, H., Dekhtyar, M., Gregory, M., Guan, X., Gupta, J., Han, J., Hargrove, A., Ho, S., Johnson, T., Legaspi, R., Lovett, S., Maduro, Q., Masiello, C., Maskeri, B., McDowell, J., Montemayor, C., Mullikin, J., Park, M., Riebow, N., Schandler, K., Schmidt, B., Sison, C., Stantripop, M., Thomas, J., Thomas, P., Vemulapalli, M., Young, A., Mullikin, J., Gnanakaran, S., Hraber, P., Wiehe, K., Kelsoe, G., Yang, G., Xia, S., Montefiori, D., Parks, R., Lloyd, K., Scearce, R., Soderberg, K., Cohen, M., Kamanga, G., Louder, M., Tran, L., Chen, Y., Cai, F., Chen, S., Moquin, S., Du, X., Joyce, M., Srivatsan, S., Zhang, B., Zheng, A., Shaw, G., Hahn, B., Kepler, T., Korber, B., Kwong, P., Mascola, J., & Haynes, B. (2013). Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus Nature, 496 (7446), 469-476 DOI: 10.1038/nature12053

Wallflowers!

I'm thrilled to announce that four of my flowers will be displayed at the Fuller Lodge Art Gallery in Los Alamos, NM, from June 14 until July 27th in a collective titled "Wallflowers." Lots of other artists from NM will be there, so check-out the link in a few days and you'll be able to see a preview of their work. These are mine:

Can't make it to NM this summer? No worries, you get a second chance to see my work in the fall: yes, I will have my very first gallery show in Santa Fe, NM, this coming fall: from September 27 until October 8th my flowers and sunsets will be at the Silver Sun Gallery on Canyon Road, one of the most characteristic roads in Santa Fe. Hope you can make it then and stay a few more days for Balloon Fiesta in Albuquerque! :-)

Bug hunting and other NM encounters

Whenever I put the macro lens on my camera and set out for a field trip I learn something new. Last year I found a metallic green sweat bee and a yellow bee with blue eyes. This year I found a black bee with green eyes and ...

A yellow heart bug (yes, I know, none of these are scientific names, in fact, if somebody can provide a scientific name, please do so in the comments, I'll edit them in):

A golden bee:

And (drum roll, please, because I think this one's the star of the show) a metallic blue bee:

EDIT: thanks to Steven Halter's attentive eye, this beautiful bee has been identified as Osmia ribifloris. Thanks, Steve! Here she is, again, poking her head out of the thistle thorn and realizing she's no longer alone. "Ladies! I was here first!"

Bugs weren't the only encounters. This fellow gave us a lesson on where to properly cross the road. A huge SUV politely stopped and waited.

Finally, I did swap lenses at some point and took this picture for my friend Hollis, who's the expert on rock formations and their awesome colors. These are red rocks in Jemez Springs, NM.

Happy Memorial Day every one! :-)

Should you worry about vitamin D deficiency? Maybe. Or maybe not.

Since my last blog post, where I shared my thoughts on BRCA1, BRCA2, and preventive mastectomies, I've been asked what else can a woman do to reduce her risk of breast cancer. I've heard a big deal about vitamin D, so I did a bit of research on the matter.

As a disclaimer, I should tell you up front that, though many correlations between vitamin D deficiency and cancer risk have been found, just as many have been refuted or found inconclusive. You can read more about it on the wikipedia page.

What is vitamin D?

The name "vitamin D" includes a group of steroid-like molecules (they are similar to steroids, but not quite steroids) that help our intestine absorb calcium and phosphates. Since calcium is essential in bone development, vitamin D deficiency has been most commonly associated to osteoporosis and other bone-related diseases. There aren't many foods rich in vitamin D, however, vitamin D can be endogenously synthesized when the skin is exposed to sunlight. Unfortunately, modern lifestyle keeps us cooped up many hours in office cubicles, or in the house during chores, or in malls. When we're out enjoying the sunshine we cover up with hats and super-protective sunscreens because we've been told that the sun is bad for the skin and can cause malignancies. As a consequence, vitamin D deficiency is increasing world-wide.

There is a foundation for all the studies that have analyzed correlations between several diseases, including cancers, and vitamin D: (i) several ecological studies have found a trend for an increase in incidence of certain cancers at higher latitudes, suggesting that longer exposures to the sun may have a protective effect. (ii) The vitamin D receptor (VDR) is expressed in many cells of the immune system, and mouse models have shown that vitamin D deficiency can promote certain auto-immune diseases. In a recent review, Sundaram and Coleman examine the link between vitamin D and influenza [Adv. Nutr. 2012 3: 517-525]. (iii) "VDR regulates a wide range of cellular mechanisms central to cancer development, such as apoptosis (cell death), cell proliferation (uncontrolled cell growth), differentiation, angiogenesis, and metastasis [1]". In line with this observation, Pereira, Larriba, and Munoz published a review on the evidence that vitamin D plays a protective role in colon cancer [Endocr. Relat. Cancer 2012 19: R51-R71].

In [1], Crew discusses the use of vitamin D supplementation as part of breast cancer prevention. She presents many interesting findings, for example:

"Colon, breast, and lung cancer have all demonstrated downregulation of expression of VDR when compared to normal cells and well-differentiated tumors have shown comparably more VDR expression as measured by immunohistochemistry when compared to their poorly differentiated counterparts. Higher tumor VDR expression has also been correlated with better prognosis in cancer patients [1]."

Crew looks at different types of studies: some suggest beneficial effects from using vitamin D (calcitriol) in combination with other anti-cancer treatments; some found an inverse association with mammography density, a biomarker for breast cancer (supposedly high density increases the risk of cancer); some found an inverse association between better breast cancer prognosis and vitamin D deficiency. However, many of these studies have limitations. For example, some only assess the levels of vitamin D through dietary intake, which is not a good measure of the circulating levels because it doesn't account for vitamin D synthesized through sun exposure. Some were confounded by obesity since fat is known to sequestrate vitamin D and also raise breast cancer risk. In light of all these considerations, Crew concludes:

"Even with substantial literature on vitamin D and breast cancer, future studies need to focus on gaining a better understanding of the biologic effects of vitamin D in breast tissue. Despite compelling data from experimental and observational studies, there is still insufficient data from clinical trials to make recommendations for vitamin D supplementation for breast cancer prevention or treatment [1]."

As I often do in my posts, rather than giving you answers, I make an effort to provide you with pointers and food for thought: in the end you have to make your own decisions about your health and the wellbeing of your family. As a personal note, I'll add that on my last blood report my vitamin D circulating levels were undetectable. I had no symptoms whatsoever, but I am now taking a vitamin D supplement. I'm also much less paranoid about smothering my kiddos with sunscreen when they play outside (which has made them much happier, two birds with one stone).

[1] Crew, K. (2013). Vitamin D: Are We Ready to Supplement for Breast Cancer Prevention and Treatment? ISRN Oncology, 2013, 1-22 DOI: 10.1155/2013/483687

Angelina no longer has them. Does that mean I should get rid of them too?

We love them and yet we hate them. They get censored, augmented, reduced, replaced, covered, exposed. They get grilled, occasionally, but those are not the ones I'm talking about. We want to see them and yet we pretend we don't. We criticize them and yet we forget what they are made for, the most beautiful thing of all: nourish a new life.

Yes, I'm talking about breasts.

Angelina Jolie's breasts have been extensively discussed this week, more now that they are reportedly gone than when they were around. Sort of ironic, if you thin about it. Angelina did the unthinkable: she had both her healthy breasts removed to prevent cancer. In a second phase of her preventive plan, she will have her ovaries removed, too. The tabloids will no longer be able to speculate on her possible new pregnancies, but they will have plenty to discuss on and around her missing body parts.

Somehow the news left me a little puzzled, unable to share the views of those who praised Angelina for her bravery. Yes, it takes guts to do what she did. At the same time, the huge resonance she's been given seems blown out of proportion. Just another Hollywood thing. It reminds me of back when our mothers were told that formula was way better than breast milk. Are we facing a new era where silicon is better than milk ducts? Are they trying to convince us that fake is healthier than real? Well, of course it is. It's fake!

So, before we go around demonizing breasts and invoking chopping off body parts in the name of longevity, I wanted to get some facts straight.

First of all, I read over and over again, "Angelina Jolie carries the gene BRCA1 ..." Turns out, we all carry the gene. What makes us different is that there are distinct copies of this gene across individuals, and some copies (but not all) do raise the risk of breast and ovarian cancer.

BRCA1 and BRCA2 are part of the so called tumor suppressor genes, genes that code for proteins that are in charge of repairing damaged DNA. Our cells undergo numerous cellular divisions during our lifespan, and every cell division carries a certain chance of damaging the DNA. Though rare, mutations can be introduced, which can either be lethal or create a cancerous cell. Tumor suppressor proteins make a first attempt to repair the damaged DNA. If the DNA cannot be repaired, they promote apoptosis, or cell death. Another example of tumor suppressor gene is TP53, which encodes the protein p53.

The first link between BRCA1 and breast cancer was discovered in 1990 by Hall et al. [1]. BRCA1 and BRCA2 are expressed mostly in breast tissue. Some mutations in these genes cause them to code proteins that are not fully functional. When this happens, a cell with damaged DNA has a higher chance to escape the "screening" and start dividing instead of undergoing apoptosis. Because BRCA1 and BRCA2 are expressed mostly in the breast tissue, by removing the breast tissue one gets rid of the majority of cells expressing the defective genes, which in turns significantly lowers the chance of developing breast cancer.

While hundreds of mutations/variations in the BRCA1 and BRCA2 genes have been found, not all are linked to breast cancer, and the ones that are don't increase the risk in the same amount. Furthermore, the majority of breast cancers are not linked to mutations in these two genes. In other words, having the mutations raises the risk, but not having them does not lower it.

So, let's get some numbers straight. According to the American Cancer Society about 15% of women diagnosed with breast cancer have a family member diagnosed with it. That leaves the majority of breast cancers unrelated to family history:

"About 85% of breast cancers occur in women who have no family history of breast cancer. These occur due to genetic mutations that happen as a result of the aging process and life in general, rather than inherited mutations."

It's a puzzle I've discussed before, the missing herediatbility. On the one hand we know genes play a large role in cancer and we spend all this research money into looking for genetic causes. Yet, the vast majority of cancers are non-hereditary.

While women with certain mutations in either the BRCA1 or BRCA2 genes have up to 80% (the exact chance varies depending on the type of mutation they carry) increased risk of developing breast cancer, only between 5% and 10% of breast cancers are linked to deleterious mutations in the BRCA1 or BRCA2 genes. So, yes, get tested. But chances are, your copy of BRCA1 and BRCA2 are fine.

So, what makes BRCA1 and bRCA2 so scary?

The American Cancer Society reports that approximately 60% of women with one of the harmful mutations in BRCA1 or BRCA2 develop breast cancer during their lifetime, versus the 12% of women in the general population. Remember, though: these genes are not the only ones playing a role in cancer. Things like epistasis with other loci in the genome can deeply affect such risks and, unfortunately, we still don't know enough to quantify them. High levels of IGF-1, the insulin-like growth factor have also been linked to breast cancer. So while having those mutations raises the risk, it does not mean that the individual will develop breast cancer for sure as other factors are still unknown. Careful considerations should be made before making a drastic choice like Angelina's. These considerations should also include risks associated to a double mastectomy (infection, necrosis, etc.) and reconstruction surgery, neither one free of complications. I'm somehow reluctant to consider implants healthier than normal breasts, whether or not those breasts were expressing faulty genes.

What about those 85% of breast cancers that are not linked to BRCA1 or BRCA2 mutations? Can we do anything to prevent those?

When you look at the global population, the most common risk factors for breast cancer are not the mutations in BRCA1 and BRCA2, rather, as Bernstein reports in a 2009 review [2]:

"The most consistently acknowledged risk factors for breast cancer other than gender and race/ethnicity are age, family history of breast cancer, early menarche, late age at first birth, nulliparity, late age at menopause, high postmenopausal weight or substantial weight gain as an adult, exposure to high levels of ionizing radiation and a history of benign proliferative breast disease [2]."

All these risk factors point at one common etiology, ovarian hormones (estradiol and progesterone), because they

"promote cellular proliferation in the breast, providing greater opportunity for the accumulation of random errors, which may lead to tumor development [2]."

Body weight and exercise can be linked to different levels of estradiol in the blood (high body weight is associated with higher levels, exercise is associated with lower levels), hence their correlation to breast cancer risk. Some studies found up to 40% reduction in risk in women who exercised in particular in their adolescence. Of all risk factors, these two, body weight and exercise, are the ones we can actually take control over and actively lower our risk of developing breast cancer. A diet rich in antioxidants may lower the risk of DNA damage during cellular division.

Things we have less control over is the woman's age at the first pregnancy. One of my grad school professors used to say, "Having a baby as a teen may ruin your life, but it sure lowers your risk of developing breast cancer later in life." The risk keeps lowering for every additional pregnancy, though not as significantly as with the first one.

What's not clear is the extent to which breastfeeding can lower the risk of breast cancer, as the American Cancer Society reports:

"Research suggests that breastfeeding has only a slight effect on breast cancer risk and that effect is only among women who have breastfed for a long time. They also concluded that breastfeeding seems to be more protective against the most aggressive types of breast cancer, including tumors in women with mutations in the BRCA1 gene, basal-like cancers, hormone-receptor negative, and possibly triple negative tumors."

And while we do the things that we can to lower our risks, I am hopeful that one day gene therapy will be perfected to the point that it will offer a better options than what, in gross terms, amounts to amputation.

Thoughts?

[1] Hall, J., Lee, M., Newman, B., Morrow, J., Anderson, L., Huey, B., & King, M. (1990). Linkage of early-onset familial breast cancer to chromosome 17q21 Science, 250 (4988), 1684-1689 DOI: 10.1126/science.2270482

[2] Bernstein, L. (2008). Identifying population-based approaches to lower breast cancer risk Oncogene, 27 DOI: 10.1038/onc.2009.348

Pumping fuel from bacteria

In my last post I discussed a bioengineered E. coli strain capable of producing an engine compatible biofuel. I hailed the finding as more efficient than ordinary biofuels because this technique has less environmental impact than biofuels from crops, for example, or cellulose, which instead use great amounts of water and forest land.

I did some more reading on the topic and found out that, surprise surprise, there are some costs in harvesting biofuels from bacteria as well, so my discussion was incomplete. However, there are good news at the horizon.

When I first read the Howard et al. paper, I imagined a petri dish of E. coli sitting in a slime of oil-like substance. I think I got confused with making yogurt. :-) In reality, the biofuel molecules are stored inside the cells (bacteria, in this case) and need to be taken out without harming the cells. Biofuel secretion strategies have been dubbed "milking." The difference, though, is that contrary to milk and cows, biofuels are generally toxic to the bacteria that produce them.

Several methods have been investigated to efficiently "milk" biofuel molecules out of bacteria without harming them. To understand these strategies, we need to learn a new concept: an efflux pump is a membrane transporter protein that carries a substance toxic to the cell outside the cell itself. These proteins remove all kinds of toxic substances, including antibiotics, for example, and they may be specific to one in particular, or carry a whole range.

In [1], Dunlop et al. discuss the use of efflux pumps in "milking" biofuels out of bacteria and reduce their toxicity to the cells:

"Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity."

The researchers first looked at the whole genome of E. coli to identify all genes encoding efflux pumps. They found 43 different pumps expressed in the E. coli genome, and tested them against a range of possible biofuels. Their strategy was as follows: the grew a culture of pooled bacteria with different subpopluations, each subpopulation expressing a different pump. In the absence of toxic biofuel-like substances, all subpopulations grew in equal proportions, and none had an advantage over the others. When a substance was introduced, the subpopulations with the most advantageous pumps with respect to that particular substance outgrew the rest.

This is what happened, for example, when they introduced geranyl acetate:

"When the pooled culture was grown in the presence of an inhibitory biofuel such as geranyl acetate, some efflux pumps conferred a distinct advantage. Although all strains started out with equal representation, after 38 h the population composition changed, with cells containing the advantageous pumps becoming an increasingly large proportion of the population. The efflux pumps that enhanced tolerance to geranyl acetate originated from a variety of hosts and include both known and previously uncharacterized pumps."

In their study, Dunlop et al. used a type of membrane transporters called "RND," which are made of big molecules and are only found in Gram-negative bacteria. In a more recent paper [2], Doshi et al. studied a broader set of pumps called ABC, ATP-binding cassette:

"Unlike RND proteins, transporters belonging to the ATP- binding cassette (ABC) protein family are widely found in all five kingdoms of life. They share a conserved structural architecture and specifically import or export a wide variety of molecules and ions across cellular membranes."

Doshi et al. tested whether this family of broadly specific pumps could efficiently mediate the secretion of four different biofuel molecules. Similarly to the Howard et al. paper, they used a bioengineered strain of E. coli and noticed that

"the secretion process was sustained for at least 6 days without the need to replenish the growth medium or culture. Thus, for the same quantity of biofuel produced conventionally, we have a dramatic reduction in biomass scale and significant gain in the ease of recovering the biofuel."

Though my understanding is that work still needs to be done to improve this technique and make it feasible for different types of biofuels, the fact that these transporters are spread across different species makes it potentially translatable to other organisms and therefore of broader use.

On a completely different note, can you guess what the macro picture is? :-)

[1] Dunlop, M., Dossani, Z., Szmidt, H., Chu, H., Lee, T., Keasling, J., Hadi, M., & Mukhopadhyay, A. (2011). Engineering microbial biofuel tolerance and export using efflux pumps Molecular Systems Biology, 7 DOI: 10.1038/msb.2011.21

[2] Doshi, R., Nguyen, T., & Chang, G. (2013). Transporter-mediated biofuel secretion Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1301358110

Fill the tank, please. With bacteria!

I apologize if you've already heard about this, but the paper is really cool and I couldn't resist discussing it here.

Escherichia coli, or E. coli for brevity, is a bacterium normally associated with "bad" things like food poisoning. Even though most strains are actually harmless, even the CDC has a page dedicated to E. coli outbreaks. Since it's part of our gut flora, the lower intestines in particular, it's usually not a good sign when E. coli is found in places like restaurants and cafeterias. (Yuck!)

What's less known to the public is that E. coli is one of the most studied bacteria and makes a great model for mutations, gene duplications, and horizontal gene transfer.

What's even less known is that this amazing bacterium has the potential to save our planet from further drilling. How? By producing fuel. Yes, you read that right: through a combination of gene modifications, researchers from the University of Exeter [1] induced "petroleum-replica hydrocarbons" production in E. coli. These hydrocarbons are structurally and chemically similar to fossil fuels.

In their paper, Howard et al. argue against the current biofuels because they bring additional costs in downstream processing and are not 100% compatible with the engines on the market.

"To overcome the end-user blend wall, it is essential to generate precise chemical replacements to fossil fuels through sustainable means.Retail transport fuels are composed primarily of hydro- carbons (n-alkanes) of various carbon chain lengths (Cn), branched hydrocarbons (iso-alkanes), and unsaturated hydrocarbons (n- alkenes). The ideal biofuels are therefore n-alkanes, iso-alkanes, and n-alkenes that are chemically and structurally identical to the fossil fuels they are designed to replace [1]."

Gasoline, diesel and jet fuels are made primarily of molecules called alkanes, or saturated hydrocarbons. Most people are familiar, or at least have heard of methane, the simplest alkane molecule. These molecules are naturally produced not just by bacteria, but also by plants and insects when they metabolize fatty acids. In 2010 Schirmer et al. described in a Science paper [2] an alkane biosynthesis pathway in cyanobacteria, commonly known as blue-green algae.

"The pathway consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes [2]."

Understanding how alkanes are produced and, in particular, which genes are involved in their production, was the first step. The second step was answering the question: can we tweak this pathway to produce alkanes that can replace our current fuels?

Seen under this light, the PNAS study published last March 15 [1] is a bioengineering success story. Howard et al. designed a novel metabolic pathway that forced E. coli to use free fatty acids instead of fatty acid compounds as in cyanobacteria, and produce fuel-like alkanes, what the authors call "industrially relevant, petroleum replica fuel molecules." Once finalized, this type of biofuel will be compatible with current engines and will not need to be blended with other petroleum derived chemicals.

A bit of perspective: though derived from natural and biological sources, biofuels still contribute to pollution, carbon emissions, and global warming. Despite the amicable "bio" prefix, they all come with a non-null carbon footprint, some more than others. The true efficiency of any kind of fuel is the energy they produce minus the energy and costs it takes to derive them. For example, producing biofuels from crops drains precious resources, first and foremost, water, but also arable land, forests when arable land is not available, and food sources in underdeveloped countries.

So here's where biofuels from bacteria have a striking advantage: E. coli is one of the cheapest and easiest bacterium to grow in a lab. It doesn't drain water reservoirs and it doesn't need deforestation to grow. Contrary to most biofuels out there, that have high production and energy costs, the carbon footprint of biofuels derived from bacteria only comes from carbon emissions when you burn them.

And while this is an excellent thing, I still think that the real change we need to make to preserve our planet is to switch to renewable energy.


[1] Howard, T., Middelhaufe, S., Moore, K., Edner, C., Kolak, D., Taylor, G., Parker, D., Lee, R., Smirnoff, N., Aves, S., & Love, J. (2013). Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1215966110

[2] Schirmer, A., Rude, M., Li, X., Popova, E., & del Cardayre, S. (2010). Microbial Biosynthesis of Alkanes Science, 329 (5991), 559-562 DOI: 10.1126/science.1187936