Personal Cancer Treatment

Reference: Muller et al. “Passenger deletions generate therapeutic vulnerabilities in cancer.” Nature (2012) 488, pgs 337 – 341

Reference: Lehner and Park. “Exploiting collateral damage.” Nature (2012) 488, pgs 284 – 285 

Reference: Johnston, Iain. “The chaos within: Exploring noise in cellular biology.” Significance (2012) August, pgs 17 – 21 

                Innovative ways to specifically kill cancer cells within the context of a living human body are necessary.  The scientific community has plenty of ideas: Trojan horse proteins bearing chemotherapeutics, exploiting the nature of cancer cell surface receptors, intense high throughput studies to identify cancer-sensitive compounds and targeted therapies towards known oncoproteins.  Unfortunately, just as each human is unique, cancer is an all-encompassing term for hundreds of different diseases that each has their own set of complications to be overcome.  Forward thinking and ingenuity are keys to successful progression.  To this end, Muller et al. report in this week’s Nature magazine about an experimental design that identifies key vulnerabilities in cancer cells by highlighting what proteins are not present.

                Enolase is an essential enzyme necessary for the second to last step of glycolysis.  Three homologues of this enzyme exist with three different gene expression profiles: ENO1 is ubiquitous, ENO2 is restricted to neurons, and ENO3 is only in muscle.  It has been shown that invertebrates and mice carry several homologous genes that encode for proteins capable of doing each other’s jobs.  The beauty of this redundancy occurs when one of the genes is knocked out: the other proteins are able to pick up the slack and death isn’t an inevitable result.  In the case of enolase, both ENO1 and ENO2 are expressed in neural cells and both are capable of performing the same function.  But, imagine that one gene becomes mutated.  The cell would then have an unhealthy reliance on the other ENO gene.  Since the enzyme is essential, a blow to the other enolase homologue should result in death to that cell.

                Cancer cells have an unhealthy habit of collecting mutations.  The Cancer Genome Atlas Research Network has sought to study the genomes of cancer to establish what mutations have turned a once healthy cell into a feast of illness.  Gliobastomas are a type of brain tumor that affects glial cells, which expresses both ENO1 and ENO2, but is far more reliant on ENO1.  Interestingly, the lp36 locus, home of the ENO1 gene (among others) is often deleted in glioblastoma.  In theory, this should create an Achilles Heel out of ENO2. 

                Muller et al. began with two different cells lines: one expressed both ENO1 and ENO2 (ENO1 wild type) while the other only expressed ENO2 (ENO1-null).  Upon two independent shRNA-mediated knockdowns, only the cells only expressing ENO2 displayed marked inhibition of proliferation.  Wanting to further prove their concept, the authors then treated the cells with the enolase inhibitor phosphonoacetohydroamate (PHAH).  The compound displayed potent toxicity towards the ENO1-null cells and little impact on the ENO1 wild type cells.  Finally, PHAH was titrated into cells with varying degrees of both ENO1 and ENO2 expression.  Intriguingly, the data showed a direct relationship between the sensitivity of the cells to PHAH and their enolase activity profiles.  

                The lp36 locus contains other essential housekeeping proteins.  In addition to these cells being reliant on ENO2, they might also be unnaturally resting on other individual proteins whose homologues have been knocked out.  Determining what they are and inhibiting them as well could lead to even greater effectiveness at cancer cell-specific death.  The paper ends up with this thought: “By one estimate, 11% of all protein-coding genes in the human genome are deleted in human cancers.  Thus, given the large number of homozygous deletions across many different cancer types spanning many hundreds of genes, the model described here for [glioblastoma] should be applicable to the development of personalized treatments for many other cancer types.”

                While reading this paper, I kept thinking back to another article I read in the journal Significance concerning the random events and complete chaos that is the inside of a cell.  Take two cells with exactly the same genome.  Variability exists in the expression profiles of all genes between those two cells due to random chance.  In the Muller et al. example, one single cancer cell in a gliobastoma may express ENO2 at a much higher level than its neighboring cell, which means that Cell A will need more drug to kill it than Cell B even though both have the exact same genome.  But scientists don’t consider Cell A and Cell B, we consider whole cell populations and assume they are all acting identically.  One IC₅₀ value represents the cells in that particular plate at that particular time and is averaged with other cell populations at different times.  Eventually, scientists are looking at averages of averages.  We make broad assumptions on broad pieces of data that are based on what is happening right then.  But cancer isn’t static; tumors within a human body are an ever evolving entity that picks up more mutations and creates more roadblocks as time goes on.  Cancer is a many-headed hydra.  

Controversial Influenza Research

REFERENCE: Imai et al. "Experimental Adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets." Nature (2012), epub ahead of print.  LINK

Amedeo Post offering more background and editorials related to this work: LINK

                This week in Nature sees the publication of the long discussed influenza research paper from the University of Wisconsin-Madison lab of Yoshihiro Kawaoka.  In a recent editorial, Kawaoka urges for publication and further research in this area by saying “[s]ome people have argued that the risks of such studies…outweigh the benefits.  I counter that H5N1 viruses circulating in nature already pose a threat because influenza viruses mutate constantly and can cause pandemics with great losses of life.  …I believe it would irresponsible not to study the underlying mechanisms.”  This published paper focuses on the hemaglutinin (HA) protein from the H5N1 virus circulating primarily in Southeast Asia that retains specificity and virulence in birds.  To date, 578 humans have become infected with this virus after direct contact with infected animals.  340 have died, but human-to-human transmission has, so far, not been an issue, but the potential for a pandemic caused by an evolving H5N1 virus is still present.  Kawaoka and colleagues sought to determine what mutations within this H5 would allow the protein to bind human receptors, if an influenza virus bearing these mutations could both efficiently infect and transmit the virus among mammals, and finally if current vaccines/antiviral therapies would be useful against such a virus.

                Human cells of the respiratory tract display α2,6-linked sialic acid with galactose while avian tracts have α2,3 linkages.  As the main receptors for HA binding, the ability of an influenza virus that was specific for birds to infect humans means, in part, that the specificity for binding switched from α2,3 to α2,6.  The authors began by introducing random mutations into the H5 globular head region where receptor binding occurs.  Turkey red blood cells were treated with sialidase to remove α2,3 linked sialic acid and preferentially leave α2,6.  Viruses were generated bearing the mutated H5s and tested for their ability to bind treated turkey red blood cells.  The identified viruses were screened again for α2,6 binding to root out false-positives and identified HAs were then further tested in solid-phase binding experiments for α2,6-specificity.  In the end, an H5 bearing mutations at E119, V152, N224 and Q226 was identified as binding only α2,6 linkages.  The authors further confirmed that N224 and Q226 mutations were critical for the shift in specificity.

                The hemaglutinin from H1N1, which was isolated from a human patient, was replaced with an H5 bearing the appropriate N224 and Q226 mutations.  Ferrets were infected with this reassortant virus and found that, after a period of 6 days, a new mutation at position N158 was observed. Viruses bearing an H5 with this new additional mutation could replicate well in ferrets and were mildly transmissible between ferrets.  Intriguingly, the viruses were found to contain yet another mutation as position T318 after ferret infection.  A new H1N1 virus bearing a quadruple H5 mutant protein was found to be highly transmissible between ferrets.  No ferrets died as a result of infection with either virus.  Encouragingly, the authors also showed that a prototype H5N1 vaccine was reactive with the mutant viruses created here and the viruses were susceptible to a licensed NA inhibitor.

                Specific mutations at N224 and Q226 are mostly likely changing the binding pocket to accommodate α2,6 linkages; a specific N158 mutation removes a glycosylation site on H5 that could be improving transmissibility.  These three mutations destabilize the protein in an acidic environment but the T318 mutation returns that stability.  Membrane fusion of the influenza virus with the host cell occurs at low pH so stability at these [H⁺] is necessary.

                It should be noted that hemaglutinin, while highly involved, is only one protein involved in the virulence of an influenza virus.  Studies show that neuraminidase also plays a role.  It was also stressed that the remaining genes in the mutant virus came from H1N1, not the avian-virulent H5N1.  It’s possible that the remaining genes in the influenza also contribute to the virulence of a virus in new hosts.  The hope is that the amino acid mutations identified here will help those keeping an eye on current H5N1 viruses.  Should any of these mutations arise, they will have the tools to predict pandemic potential and, knowing that current therapies are effective against these viruses, that proper safeguards can be put in place quickly.  As a final thought, the authors realize that a pandemic virus may not even show these mutations and something else entirely, but the work has identified important areas of the HA protein that could and most likely will change as an influenza virus evolves from avian to mammal specificity.

 

Influenza Research Pause

REFERENCE: Fouchier et al. “Pause on avian flu transmission studies.”  Nature (2012)

LINK directly to published letter

                As I discussed in my American Society for Cell Biology Meeting post, I don’t want to repeat work that has already been discussed outside of the initial scientific publication, however this topic is interesting, especially considering the letter was signed by 39 authors and published in both Nature and Science magazines.  

                Work being performed at the University of Wisconsin-Madison and Erasmus MC in the Netherlands has suspended important research on a highly transmittable influenza virus due to fears of viral escape from their laboratories.  They have imposed a 60 day “pause” on their work while the scientific community and the community at large have time to discuss some of the new issues this type of research presents.

Evolution of Red Giants

REFERENCE: Charpinet et al. “A compact system of small planets around a former red-giant star.” Nature (2011) 480, pgs 496 – 499.    

Red giants are low to intermediate mass stars late in their lifetimes that have swelled to massive sizes.  It has been predicted that the Sun will become a red giant is 7.5 billion years at which time its radius will become 200 times larger than it is now.  Typically, planets that are orbiting a star at a radius less than 1 astronomical unit (AU) will be engulfed by the swelling red giant.  However, some post red giant stars still have giant planets orbiting them at radii much closer than 1 AU.

                Recent work by Telting and colleagues suggests both how planets can survive immersion in the red giant envelope and influence the evolution of the star.  KIC 05807616 (also known as KPD 1943+4058) is a B subdwarf star, which is much hotter and brighter than a typical subdwarf star and represents a rarer way a red giant star can evolve.  This type of subdwarf evolves when a red giant loses its outer envelope prematurely.  Charpinet et al. show that KIC 05807616 has two planets, which they name KOI 55.01 and KOI 55.02, slightly smaller than Earth still orbiting it.

                They authors further offer a reasonable scenario as to how the current system came to be.  They feel that both bodies were originally large, gaseous planets that were swallowed by the red-giant envelope.  The immersion triggered the red giant’s outer shell premature loss and evolution into a B subdwarf, however this process stripped the planets of their gaseous layers and left only the inner cores, which the authors see orbiting the star today.

                An alternative scenario involving the merging of two white dwarfs that resulted in planet formation followed by a secondary planet is possible, but highly unlikely.

The Great Sirtuin Debate

REFERENCE: Burnett et al. “Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila.” Nature (2011) 477, pgs 482 – 485.

                It all started with a report that overexpression of SIR2 in budding yeast led to increased lifespan.  Follow up studies showed similar results in C. elegans and Drosophila, which lead researchers to pursue the relationship between calorie restriction (a known way to extend lifespan) and sirtuin expression.  These results bore resveratrol, a purported activator of human Sirtuin 1 (SirT1), which most of the general public will tell you is a component of red wine.  

                The dissent on the role of resveratrol and sirtuins in lifespan extension comes from labs at the University of Washington, University of Wisconsin, Amgen Inc., and Pfizer, among others.  Their papers explicitly say resveratrol has no activating properties on SirT1, conclusions which they back up with control studies using the Fleur-de-Lys system and crystal structures.

                Another blow to the importance of sirtuins came in a recent issue of Nature.    Burnett et al. studied C. elegans and Drosophila overexpressing sir-2.1 and closely accounted for the genetic backgrounds of each.  When taking into account these parameters, longevity increase was no longer noted.  Within fruit flies, the authors further concluded that dietary restriction did increase fly lifespan but was not dependent on Drosophila Sir2.  

Gem and colleagues stress the importance of “…controlling for genetic backgrounds and for the mutagenic effects of transgene insertions in studies on genetic effects on lifespan.”  As for the importance of sirtuins, they cannot support a strong relationship between sirtuins and lifespan extension.