UPenn Scientists Are Investigating Better Treatments for Sarcoma Tumors

by Adrian Rivera-Reyes and Koreana Pak

Soft tissue sarcomas (STS) are rare cancers of the connective tissues, such as bone, muscle, fat, and blood vessels. Soft and elastic, sarcoma tumors can push against their surroundings as they grow silent and undetected. Residing in an arm, torso, or thigh, it can take years before a sarcoma begins to cause pain. By the time a patient presents their tumor to a doctor, amputation may be unavoidable¹.

In 2017, it is predicted that 12,390 Americans will be diagnosed with sarcoma, and approximately 5,000 patients will die from these tumors². But the vast majority of these patients aren’t dying from the first tumor in their arm or leg—the real danger is metastasis, which is responsible for more than 90% of cancer-related deaths^3-5.

Metastasis occurs when tumor cells leave their original site and colonize a new area of the body, such as the lungs, liver, or bones^3-5. The current treatment options for sarcoma—surgery, chemotherapy, and radiation—are not very effective against metastases^6,7. Only 10-25% of STS patients respond to chemotherapy, leaving surgery as the best option for many^6,7. However, tumor cells can spread to other parts of the body even in early stages of sarcoma, long before the first tumor is even noticed. By the time the tumor is surgically removed, metastases have usually developed in other parts of the body.

As a sarcoma tumor grows, it becomes increasingly starved of oxygen and nutrients. Under these conditions, cancer cells are driven to metastasize. Moreover, tumor hypoxia, or low oxygen levels, are an important predictor of metastasis and low survival in sarcoma patients^8-10. In other words, the more tumor hypoxia, the lower a patient’s chance of surviving.

But how does this actually work? How does hypoxia drive sarcoma cells out of a tumor and into other organs, such as the lungs? Surprisingly, UPenn scientists have found it has a lot to do with collagen¹¹!

Metastasizing tumor cells (pink) associated with collagen (blue). Image taken by Koreana Pak.

Collagen is the most abundant protein in the human body, but you’ll know it best as the substance that makes your skin flexible and elastic¹². This elastic material has many uses, and you can find it in gelatin, marshmallows, surgical grafts—and hypoxic tumors. In STS tumors, the low oxygen levels cause collagen to form sticky, tangled fibers.  Sarcoma cells will actually hijack this disorganized collagen and use it as a “highway” over which they can migrate out of the tumor and into other organs¹¹.

If these hypoxic collagen “highways” were disrupted in patient tumors, cancer cells could be prevented from metastasizing. But how?

In an effort to make this therapy a reality, UPenn scientists used models of human sarcoma and metastasis in which they could disrupt collagen. By deleting the hypoxia factors HIF-1 and PLOD2, they could restore normal collagen in tumors, which reduced tumor metastasis. Excitingly, they also found that minoxidil, a drug usually used to treat hair-loss, also reduced tumor collagen and halted metastasis¹¹.

Whether minoxidil could be used for human patients is unclear; nevertheless, drugs that reduce hypoxic targets like PLOD2 could serve as promising anti-metastatic therapies.

In a follow up study, these scientists looked at another hypoxic factor, called HIF-2¹³. While related to HIF-1, this protein actually plays a very different role in sarcoma. Elimination of HIF1 is important because it reduces metastasis¹¹. But when it comes to primary sarcoma tumors, the expression of HIF-2 can help reduce cancer cell growth¹³.

Again using a model of human sarcoma, the authors found they could increase tumor size when they eliminated HIF-2. They also used a clinically approved drug, Vorinostat, to treat these tumors, and saw that HIF-2 increased and as a consequence the tumors to shrank¹³.

Sarcoma Treatment: Going Forward

The diversity of STS, which comprises about 50 different types¹, as well as the low incidence of cases, makes it very challenging to develop better treatments for sarcoma. Clinical trials often combine patients with different types of sarcomas into a single study, even though the trial may not be a good fit for all the patients. A more specific approach is needed to treat the different types of sarcomas.

Through their research on hypoxia in sarcoma, UPenn scientists hope to improve current treatments. Their observation that HIF-1 and HIF-2 play opposing roles in different cancers is of particular importance, because HIF inhibitors are already being developed for cancer therapy^11,13. Doctors can also use markers like HIF-2 to predict how well patients will respond to different treatments. For example, patients with tumors that have low levels of HIF-2 will respond well to treatments with Vorinostat. Unfortunately, such predictive markers are rare in STS, and the identification of additional markers should complement the development of new treatments.

Complementing standard chemotherapy with new sarcoma-specific therapies would greatly improve current treatment options. However, treating the primary tumor alone is not sufficient, as metastasis remains primarily responsible for patient death^6,7. For this reason, further study into HIF-1/PLOD2 and the role of collagen in metastasis is needed. Through the development of drugs like minoxidil, which target harmful tumor collagen, we see exciting potential for the future of sarcoma therapy and patient survival.

References

1. Cancer.Net Editorial Board. (2012, June 25). Sarcoma, Soft Tissue – Introduction. Retrieved on April 4, 2017 from: http://www.cancer.net/cancer-types/sarcoma-soft-tissue/introduction

2. The American Cancer Society medical and editorial content team. (2017, January 6). What Are the Key Statistics About Soft Tissue Sarcomas? Retrieved on April 4, 2017 from https://www.cancer.org/cancer/soft-tissue-sarcoma/about/key-statistics.html

3. Mehlen, P., & Puisieux, A. (2006). Metastasis: a question of life or death. Nature Reviews Cancer, 6, 449-458.

4. Monteiro, J. & Fodde, R. (2010). Cancer stemness and metastasis: therapeutic consequences and perspectives. European Journal of Cancer, 46 (7), 1198-1203.

5. Nguyen, D.X., Bos, P.D., & Massagué, J. (2009). Metastasis: from dissemination to organ-specific colonization. Nature Reviews Cancer, 9, 274-284.

6. Linch, M., Miah, A. B., Thway, K., Judson, I. R., & Benson, C. (2014). Systemic treatment of soft-tissue sarcoma-gold standard and novel therapies. Nat. Rev. Clin. Oncol. 11(4), 187-202.

7. Lorigan, P., Verweij, J., Papai, Z., Rodenhuis, S., Le Cesne, A., Leahy, M.G., Radford, J.A., Van Glabbeke, M.M., Kirkpatrick, A., Hogendoom, P.C., & Blay, J.Y. (2007). Phase III trial of two investigational schedules of ifosfamide compared with standard-dose doxorubicin in advanced or metastaic soft tissue sarcoma: a European Organization for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group Study. Journal of Clinical Oncology 25 (21), 3144-3150.

8. Shintani, K., Matsumine, A., Kusuzaki, K., Matsubara, T., Santonaka, H., Wakabayashi, T., Hoki, Y., & Uchida, A. (2006). Expression of hypoxia-inducible factor (HIF)-1 alpha as a biomarker of outcome in soft-tissue sarcoma. Virchows Arch. 449 (6), 673-681. 

9. Nordsmark, M., Alsner, J., Keller, J., Nielsen, O.S., Jensen, O.M., Horsman, M.R., & Overgaard, J. (2001). Hypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutations. Br. J. Cancer 84 (8), 1070-1075. 

10. Rajendran, J.G., Wilson, D.C., Conrad, E.U., Peterson, L.M., Bruckner, J.D., Rasey, J.S., Chin, L.K., Hofstrand, P.D., Grierson, J.R., Eary, J.F., & Krohn, K.A. (2003). [(18)F]FMISO and [(18)F]FDG PET imaging in soft tissue sarcomas: correlation of hypoxia, metabolism, and VEGF expression. Eur. J. Nucl. Med. Mol. Imaging, 30 (5), 695-704.

11. Eisinger-Mathason, T.S.K., Zhang, M., Qiu, Q., Skuli, N., Nakazawa, M..S., Karakasheva, T., Mucaj, V., Shay, J.E., Stangenberg, L., Sadri, N., Puré, E., Yoon, S.S., Kirsch, D.G., & Simon, M.C. (2013). Hypoxia dependent modification of collagen networks promotes sarcoma metastasis. Cancer Discovery, 3 (10), 1190-1205.

12. What is collagen? Retrieved on April 4, 2017 from http://www.vitalproteins.com/what-is-collagen.

13. Nakazawa, M.S., Eisinger-Mathason, T.S., Sadri, N., Ochocki, J.D., Gade, T.P., Amin, R.K., & Simon, M.C. (2016). Epigenetic re-expression of HIF-2 alpha suppresses soft tissue sarcoma growth. Nature Communications, 7, 10539

Tracing the ancestry and migration of HIV/AIDS in America

by Arpita Myles

Acquired immunodeficiency syndrome or AIDS is a global health problem that has terrified and intrigued scientists and laypeople alike for decades. AIDS is caused by the Human Immunodeficiency Virus, or HIV, which is transmitted through blood, semen, vaginal fluid, and from an infected mother to her child ^[1]. Infection leads to failure of the immune system, increasing susceptibility to secondary infections and cancer, which are mostly fatal. Considerable efforts are being put into developing prophylactic and therapeutic approaches to tackle HIV-AIDS, but there is also interest in understanding how the disease became so wide-spread. With the advent of the Ebola and Zika viruses in the last couple of years, there is a renewed urgency in understanding the emergence and spread of viruses in the past in order to prevent those in the future. The narrative surrounding the spread of HIV has been somewhat convoluted, but a new paper in Nature by Worobey et. al, hopes to set the record straight ^[2].

Humans are supposed to have acquired HIV from African chimpanzees- presumably as a result of hunters coming in contact with infected blood, containing a variant of the virus that had adapted to infect humans. The earliest known case of HIV in humans was detected in 1959 in Kinshasa, Democratic Republic of the Congo, but the specific mode of transmission was never ascertained ^[3].

There has been little or no information about how HIV spread to United States, until now. HIV incidences were first reported in the US in 1981, leading to the recognition of AIDS ^[4]. Since the virus can persist for a decade or more prior to manifestation of symptoms, it is possible that it arrived in the region long before 1981. However, since most samples from AIDS patients were collected after this date, efforts to establish a timeline for HIV’s entry into the states met with little success. Now, researchers have attempted to trace the spread of HIV by comparing genetic sequences of contemporary HIV strains with blood samples from HIV patients dating back to the late 1970’s ^[2]. These samples were initially collected for a study pertaining to Hepatitis B, but some were found to be HIV seropositive. This is the first comprehensive genetic study of the HIV virus in samples collected prior to 1981.

The technical accomplishment of this work is significant as well. In order to circumvent the problems of low amounts and extensive degradation of the viral RNA from the patient samples, they developed a technique they call “RNA jackhammering.”  In essence, a patient’s genome is broken down into small bits and overlapping sequences of viral RNA are amplified. This enables them to “piece together” the viral genome, which they can then subject to phylogenetic analysis.

Using novel statistical analysis methods, Worobey et al. reveal that the virus had probably entered New York from Africa (Haiti) during the 1970s, whereupon it spread to San Francisco and other regions. Upon analyzing the older samples, the researchers found that despite bearing similarities with the Caribbean strain, the strains from San Francisco and New York samples differed amongst themselves. This suggests that the virus had entered the US multiple, discreet times and then began circulating and mutating. Questions still remain regarding the route of transmission of the virus from Haiti to New York.

The relevance of this study is manifold. Based on the data, one can attempt to understand how pathogens spread from one population to another and how viruses mutate and evolve to escape natural immunity and engineered therapeutics. Their molecular and analytical techniques can be applied to other diseases and provide valuable information for clinicians and epidemiologists alike. Perhaps the most startling revelation of this study is that contemporary HIV strains are more closely related to their ancestors than to each other. This implies that information derived from ancestral strains could lead to development of successful vaccine strategies.

Beyond the clinic and research labs, there are societal lessons to be learned as well. Published in 1984, a study by CDC (Center for Disease Control) researcher William Darrow and colleagues traced the initial spread of HIV in the US to Gaétan Dugas- a French Canadian air steward. Examination of Dugas’s case provided evidence linking HIV transmission with sexual activity. Researchers labeled Dugas as “Patient O”, as in “Out of California” ^[5]. This was misinterpreted as “Patient Zero” by the media- a term still used in the context of other epidemics like flu and Ebola. The dark side of this story is that Dugas was demonized in the public domain as the one who brought HIV to the US. As our understanding of the disease and its spread broadened, scientists and historians began to discredit the notion that Dugas played a significant role. However, scientific facts were buried beneath layers of sensationalism and hearsay and the stigma remained.

Now, with the new information brought to light by Worobey’s group, Dugas’s name has been cleared. Phylogenetic analysis of Dugas’s strain of HIV was sufficiently different from the ancestral ones, negating the possibility that he initiated the epidemic.

The saga in its entirety highlights the moral dilemma of epidemiological studies and the extent to which the findings should be made public. Biological systems are complicated, and while narrowing down origin of a disease has significance clinical relevance, we often fail to consider collateral damage. The tale of tracking the spread of HIV is a cautionary one; scientific and social efforts should be focused more on resolution and management, rather than on vilifying unsuspecting individuals for “causing” an outbreak.

References:

1. Maartens G, Celum C, Lewin SR. HIV infection: epidemiology, pathogenesis, treatment, and prevention. Lancet. 2014 Jul 19;384(9939):258-71.

2. Worobey M, Watts TD, McKay RA et al., 1970s and 'Patient 0' HIV-1 genomes illuminate early HIV/AIDS history in North America. Nature. 2016 Oct 26. doi: 10.1038/nature19827.

3. Faria NR, Rambaut A et al., HIV epidemiology. The early spread and epidemic ignition of HIV-1 in human populations. Science. 2014 Oct 3;346(6205):56-61.

4. Centers for Disease Control (CDC). Pneumocystis pneumonia--Los Angeles. MMWR Morb Mortal Wkly Rep. 1981 Jun 5;30(21):250-2.

5. McKay RA. “Patient Zero”: The Absence of a Patient’s View of the Early North American AIDS Epidemic. Bull Hist Med. 2014 Spring: 161-194.

New Research shows how to make Human Stem Cell Lines divide equally

by Amaris Castanon

For the first time, scientists have generated haploid embryonic stem (ES) cell lines in humans, as published in Nature. This could lead to novel cell therapies for genetic diseases – even color blindness (Benvenisty et al., 2016)

The study was performed by scientists from the Hebrew University of Jerusalem(Israel) in collaboration with Columbia University Medical Center (CUMC) and the New York Stem Cell Foundation (NYSCF).

The newly derived pluripotent, human ES cell lines demonstrated their ability to ‘self-renew’ while maintaining a normal haploid karyotype (i.e. without chromosomal breakdown after each generation) (Benvenisty et al., 2016).

While gamete manipulation in other mammalian species has yielded several ES cell lines (Yang, H. et al., Leeb, M. & Wutz, A.), this is the first study to report human cells capable of cell division with merely one copy of the parent’s cell genome (Benvenisty et al., 2016).

The genetic match between the stem cells and the egg donor may prove advantageous for cell-based therapies of genetic diseases such as diabetes, Tay-Sachs disease and even color blindness (Elling et al., 2011).

—

Mammalian cells are considered diploid due to the fact that two sets of chromosomes are inherited: 23 from the father and 23 from the mother (a total of 46) (Wutz, 2014; Yang H. et al., 2013). Haploid cells contain a single set of 23 chromosomes and arise only as post-meiotic germ cells (egg and sperm) to ensure the right number of chromosomes end up in the zygote (embryo) (Li et al., 2014; Elling et al., 2011).

Other studies performed in an effort to generate ES cells from human egg cells reported generating solely diploid (46 chromosome) human stem cells, which is a problem (Leeb, M. et al., 2012; Takahashi, S. et al., 2014). This study, however, reported inducing cell division in unfertilized human egg cells (Benvenisty et al., 2016).

The DNA was labeled with a florescent dye prior to isolating the haploid stem cells and scattering (the haploid cells or the cells) among the larger pool of diploid cells. The DNA staining demonstrated that the haploid cells retained their single set of chromosomes, while differentiating to other cell types including nerve, heart, and pancreatic cells demonstrates their ability to give rise to cells of different lineage (pluripotency) (Benvenisty et al., 2016).

Indeed, the newly derived haploid ES cells demonstrated pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature (Benvenisty et al., 2016).

In addition, the group of researchers successfully demonstrated usage of their newly derived human ES cells as a platform for loss-of-function genetic screening. Therefore, elucidating the genetic screening potential of targeting only one of the two copies of a gene.

These findings may facilitate genetic analysis in the future by allowing an ease of gene editing in cancer research and regenerative medicine.

This is a significant finding in haploid cells, due to the fact that detecting the biological effects of a single-copy mutation in a diploid cell is difficult. The second copy does not contain the mutation and therefore serves as a ‘backup’ set of genes, making it a challenge for precise detection.

The newly derived haploid ES cells will provide researchers with a valuable tool for improving our understanding of human development and genetic diseases.

This study has provided scientists with a new type of human stem cell that will play an important role in human functional genomics and regenerative medicine.

References:

Derivation and differentiation of haploid human embryonic stem cells. Sagi I, Chia G, Golan-Lev T, Peretz M, Weissbein U, Sui L, Sauer MV, Yanuka O, Egli D, Benvenisty N. Nature. 2016 Apr 7;532(7597):107-11.

Elling, U. et al. Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell Stem Cell 9, 563–574 (2011).

Leeb, M. et al. Germline potential of parthenogenetic haploid mouse embryonic stem cells. Development 139, 3301–3305 (2012)

Leeb, M. & Wutz, A. Derivation of haploid embryonic stem cells from mouse embryos.Nature 479, 131–134 (2011)

Li, W. et al. Genetic modification and screening in rat using haploid embryonic stem cells. Cell Stem Cell 14, 404–414 (2014).

Takahashi, S. et al. Induction of the G2/M transition stabilizes haploid embryonic stem cells. Development 141, 3842–3847 (2014)

Wutz, A. Haploid mouse embryonic stem cells: rapid genetic screening and germline transmission. Annu. Rev. Cell Dev. Biol. 30, 705–722 (2014).

Yang, H. et al. Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149, 605–617 (2012)

Penn Science Spotlight: Learning how T cells manage the custom RNA business

Chris Yarosh

This Science Spotlight focuses on the research I do here at Penn, the results of which are now in press at Nucleic Acids Research¹. You can read the actual manuscript right now, if you would like, because NAR is “open access,” meaning all articles published there are available to anyone for free. We’ve talked about open access on this blog before, if you’re curious about how that works. 

First, a note about this type of science. The experiments done for this paper fall into the category of “basic research,” which means they were not designed to achieve an immediate practical end. That type of work is known as “applied” research. Basic research, on the other hand, is curiosity-driven science that aims to increase our understanding of something. That something could be cells, supernovas, factors influencing subjective well-being in adolescence, or anything else, really. This isn’t to say that basic research doesn’t lead to advances that impact people’s lives; quite the opposite is true. In fact, no applied work is possible without foundational basic work being done first. Rather, the real difference between the two categories is timeline and focus: applied research looks to achieve a defined practical goal (such as creating a new Ebola vaccine) as soon as possible, while basic research seeks to add to human knowledge over time. If you’re an American, your tax dollars support basic research (thanks!), often through grants from the National Institutes of Health (NIH) or the National Science Foundation (NSF). This work, for example, was funded in part by two grants from the NIH: one to my PhD mentor, Dr. Kristen Lynch (R01 GM067719), and the second to me (F31 AG047022). More info on science funding can be found here.

Now that you've gotten your basic research primer, let's talk science. This paper is primarily focused on how T cells (immune system cells) control a process called alternative splicing to make custom-ordered proteins. While most people have heard of DNA, the molecule that contains your genes, not everyone is as familiar with the RNA or proteins. I like to think of it this way: DNA is similar to the master blueprint for a building, specifying all of the necessary components needed for construction. This blueprint ultimately codes for proteins, the molecules in a cell that actually perform life’s work. RNA, which is “transcribed” from DNA and “translated” into protein, is a version of the master blueprint that can be edited as needed for different situations. Certain parts of RNA can be mixed and matched to generate custom orders of the same protein, just as you might change a building’s design based on location, regulations, etc. This mixing and matching process is called alternative splicing (AS), and though it sounds somewhat science-fictiony, AS naturally occurs across the range of human cell types.

While we know AS happens, scientists haven’t yet unraveled the different strategies cells use to control it. Part of the reason for this is the sheer number of proteins involved in AS (hundreds), and part of it is a lack of understanding of the nuts and bolts of the proteins that do the managing. This paper focuses on the nuts and bolts stuff. Previous work2 done in our lab has shown that a protein known as PSF manipulates AS to produce an alternate version of a different protein, CD45, critical for T cell response to antigens (bits of bacteria or viruses). PSF doesn’t do this, however, when a third protein, TRAP150, binds it, although we previously didn’t know why. This prompted us to ask two major questions: How do PSF and TRAP150 link up with one another, and how does TRAP150 change PSF’s function?

My research, as detailed in this NAR paper, answers these questions using the tools of biochemistry and molecular biology. In short, we found that TRAP150 actually prevents PSF from doing its job by binding in the same place RNA does. This makes intuitive sense: PSF can’t influence splicing of targets it can’t actually make contact with, and it can't contact them if TRAP150 is gumming up the works. To make this conclusion, we diced PSF and TRAP150 up into smaller pieces to see which parts fit together, and we also looked for which part of PSF binds RNA. These experiments helped us pinpoint all of the action in one region of PSF known as the RNA recognition motifs (RRMs), specifically RRM2. Finally, we wanted to know if PSF and TRAP150 regulate other RNA molecules in T cells, so we did a screen (the specific technique is called “RASL-Seq,” but that’s not critical to understanding the outcome) and found almost 40 other RNA molecules that appear to be controlled by this duo. In summary, we now know how TRAP150 acts to change PSF’s activity, and we have shown this interaction to be critical for regulating a bunch of RNAs in T cells.

So what are the implications of this research? For one, we now know that PSF and TRAP150 regulate the splicing of a range of RNAs in T cells, something noteworthy for researchers interested in AS or how T cells work. Second, we describe a mechanism for regulating proteins that might be applicable to some of those other hundreds of proteins responsible for regulating AS, too. Finally, PSF does a lot more than just mange AS in the cell. It actually seems to have a role in almost every step of the DNA-RNA-protein pathway. By isolating the part of PSF targeted by TRAP150, we can hypothesize about what PSF might do when TRAP150 binds it based on what other sections of the protein remain “uncovered.” It will take more experiments to figure it all out, but our data provide good clues for researchers who want to know more about all the things PSF does.

A map of the PSF protein. Figure adapted from Yarosh et al., WIREs RNA 2015, 6: 351-367. doi: 10.1002/wrna.1280

Papers cited:

1.) Christopher A. Yarosh; Iulia Tapescu; Matthew G. Thompson; Jinsong Qiu; Michael J. Mallory; Xiang-Dong Fu; Kristen W. Lynch. TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells. Nucleic Acids Research 2015;

doi: 10.1093/nar/gkv816

2.) Heyd F, Lynch KW. Phosphorylation-dependent regulation of PSF by GSK3 controls CD45 alternative splicing. Mol Cell 2010,40:126–137.

Penn researchers interview HIV-positive adolescents in Botswana to better understand the factors affecting adherence to antiretroviral treatments

Of the more than three million children infected with HIV, 90% live in Africa. As HIV-positive children become adolescents, it is important that antiretroviral treatments are maintained to protect their own health, as well as to safeguard the adolescents from developing resistant strains of HIV and to prevent infection of other individuals.

HIV-positive adolescents’ adherence to these treatments has been identified as a public health challenge for Botswana. However, the assessment tools testing psychosocial factors that are likely associated with poor adherence have been developed in Western countries and their constructs may not be relevant to African contexts. A new study published in PLOS ONE by Penn researchers Elizabeth Lowenthal and Karen Glanz described the cultural adaptation of these assessment tools for Botswana.

The psychosocial assessments investigate factors that may affect adolescents’ adherence to antiretroviral treatments. As Lowenthal summarized, “one of the key reasons why adolescents with HIV have higher rates of death compared with people with HIV in other age groups is that they have trouble taking their medications regularly.”

Researchers looked at the following factors by testing 7 separate assessment scales developed with Western cohorts for their applicability to Botswanan adolescents.

• Psychological reactance- an aversion to abide by regulations that impose upon freedom and autonomy
• Perceived stigma
• Outcome expectancy- whether treatments were expected to improve health
• Consideration of future consequences- the extent to which adolescents plan for their futures rather than focusing on immediate gains
• Socio-emotional support- how adolescents receive the social and emotional support they need

The researchers interviewed 34 HIV-positive Botswanan adolescents in depth, sub grouped by age in order to talk about the factors in ways participants could understand.

The study confirmed the construct validity of some assessment tools, but highlighted four areas that caused tools to not relate to participants:

• Socio-emotional support for the adolescents mostly came from parents rather than peers.
• Denial of being HIV infected was more common than expected.
• Participants were surprisingly ambivalent about taking their medicine.

Some of the tools (psychological reactance, future consequences) required major modifications to obtain construct validity for adolescents with HVI in Botswana.The assessment tools were modified during the course of the study based on participant feedback. Future research will test the association between these modified assessment tools and HIV treatment outcomes in order to provide insight into how to best support HIV infected adolescents.

First author Lowenthal suggested that the study could inform studies of adolescent adherence to other treatments as well, stating that “questions that we are able to answer in our large cohort of HIV-positive adolescents will likely be generalizable to other groups of adolescents with chronic diseases.”

-Barbara McNutt 

Penn researchers identify novel therapeutic target for kidney cancer

Kidney cancer, also known as renal cancer, is one of the ten most common cancers in both men and women. The American Cancer Society’s most recent estimates state that of the predicted 63,920 new cases of kidney cancer this year, roughly 20% of  patients will die from the disease. By far, the most common type of kidney cancer is renal cell carcinoma (RCC). The majority of RCCs are clear cell RCCs (ccRCCs), a subtype characterized by metabolic alterations, specifically increased carbohydrate and fat storage. More than 90% of ccRCCs have been found to have mutations in the von Hippel-Lindau (VHL) tumor suppressor gene; however, kidney specific VHL deletion in mice does not induce tumorigenesis or cause metabolic changes similar to those seen in ccRCC tumors. So what additional factors are needed for ccRCC tumor formation and progression? A recent study by Penn researchers published in the journal Nature identified the rate-limiting gluconeogenesis enzyme fructose-1,6-bisphosphatase (FBP1) as a key regulator of ccRCC progression.

To better understand ccRCC progression, the study’s first author, Bo Li, a post-doctoral researcher in the lab of Dr. Celeste Simon, performed metabolic profiling on human ccRCC tumors while also analyzing ccRCC metabolic gene expression profiles. Compared to the adjacent normal kidney tissue, ccRCC tumors had increased amounts of metabolites involved in sugar metabolism and significantly lower expression of carbohydrate storage genes, including FBP1. Further investigation revealed FBP1 expression was reduced in almost all tumor samples tested (>600) and reduced FBP1 expression strongly correlated with advanced tumor stage and poor patient survival. Thus, understanding the role of FBP1 in ccRCCs could significantly impact the treatment of this disease.

How do reduced levels of FBP1 promote ccRCC tumor progression? The authors found that FBP1 depletion in ccRCC cells stimulates growth and relieves inhibition of sugar breakdown (glycolysis), which provides energy for the growing cancer cells. In addition, VHL mutations associated with ccRCCs prevent the degradation of a transcription factor that responds to decreases in oxygen, known as hypoxia-inducible factor α (HIFα), thus stabilizing it. Stabilized HIFα does not cause FBP1 depletion, but its activity is tightly regulated by FBP1. This study emphasized the importance of the interaction between HIFα and FBP1, particularly when glucose and oxygen levels are low, for the formation and progression of the ccRCC.

Why is this work so important? Little is known about how changes in cell metabolism contribute to the formation and progression of ccRCC tumors. As stated by Li, “elucidating how FBP1 impacts the altered metabolic and genetic programs of ccRCC improves our knowledge of the molecular details accompanying ccRCC progression, and identifies novel therapeutic targets for this common malignancy.” Future work may focus on identifying how FBP1 is suppressed and whether reversing FBP1 suppression could improve patient outcomes. 

-Renske Erion

Penn researchers identify neurons that link circadian rhythms with behavioral outcomes.

Our bodies evolved to alternate rhythmically through sleep and wake periods with the 24-hr cycle of the day. These “circadian rhythms” are controlled by specific neurons in the brain that act as molecular clocks. The experience of jet lag when we change time zones is the out-of-sync period before the brain’s internal clock re-aligns with the external environment.

How does this molecular clock work in the brain? Decades of research have uncovered that environmental signals, such as light, are integrated into a circadian clock by specific neurons in the brain. However, little is understood about how these circadian clock cells drive biological effects such as sleep, locomotion, and metabolism. A study by Penn researchers published earlier this year in Cell has discovered critical neural circuits linking the circadian clock neurons to behavioral outputs.

The researchers used the fruit fly Drosophila as a model organism because like humans, they also have circadian rhythms, yet they are very easy to manipulate genetically and many powerful tools exist to study the 150 circadian clock neurons in their brains. The study found that a crucial part of the circadian output network exists in the pars intercerebralis (PI), the functional equivalent of the human hypothalamus.

“Flies are normally active during the day and quiescent at night, but when I activate or ablate subsets of PI neurons, they distribute their activity randomly across the day,” describes the study’s first author, Daniel Cavanaugh, PhD, a post-doc working in the lab of Amita Sehgal, PhD. Importantly, the research showed that modulating the PI neurons lead to behavioral changes without affecting the molecular oscillations in central circadian clock neurons, indicating that the PI neurons link signals from the circadian clock neurons to behavioral outputs.

The study also showed that the PI neurons are anatomically connected to core clock neurons using a technique involving the fluorescent protein GFP. Cavanaugh explains, “The GFP molecule is split into two components, which are expressed in two different neuronal [cell] populations. If those populations come into close synaptic contact with one another, the split GFP components are able to reach across the synaptic space to reconstitute a fluorescent GFP molecule, which can be visualized with fluorescence microscopy.”

Additionally, their experiments showed that a peptide called DH44, a homolog to the mammalian corticotropin-releasing hormone, is expressed in PI neurons and important for maintaining circadian-driven behavioral rhythms.

While these new data are interesting for understanding general mechanisms of biology, they also have implications for human health and disease.

“People exposed to chronic circadian misalignment, such as occurs during shift work, show increased rates of heart disease, diabetes, obesity, cancer, and gastrointestinal disorders,” says Cavanaugh. “In order to understand the connection between circadian disruption and these diseases, we have to understand how the circadian system works to control the physiological outputs that underlie these disease processes.”

-Mike Allegrezza

Penn Science Spotlight: Manipulating gene expression in single cells

One goal of PSPG is to make science more accessible to the general public. Our first science spotlight features work by Matt Churgin and the Fang-Yen lab.

Scientists who want to understand how specific genes function in specific cells need the ability to manipulate gene expression, but there are few tools that allow us to ask questions at the single cell level. At best these tools can target populations of cells, but that’s not good enough for the developmental biologist who wants to know the fate of a particular cell within an embryo. A recent study out of Penn (Churgin et al. Genes Genomes Genetics, 2013) addresses these limitations by improving on a method that relies on heat-inducible gene expression and a continuous wave laser. This type of laser constantly bombards the specimen with heat and tends to warm up not just the target cell but the cells around it, causing the gene of interest to be expressed in the wrong place. To address this problem the authors used a pulsed infrared laser that heats up only the target cell, setting the stage for single cell experiments. The authors then demonstrated that they could use this to temporarily induce the expression of green fluorescent protein (GFP) in one specific cell. They then took it a step further and showed that they could induce permanent expression of GFP that could be passed down to daughter cells. This new technique will allow scientists to have greater spatial (which cell?) and temporal (when and how long?) control over gene expression. This will help answer questions such as how the fate of cells is genetically controlled during development.

Efficient Single-Cell Transgene Induction in Caenorhabditis elegans Using a Pulsed Infrared Laser
Matthew A. Churgin, Liping He, John I. Murray, and Christopher Fang-Yen

Genes Genomes Genetics October 2013

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