The videos below show 4 of our summer students talking about their summer projects. These videos were produced by our awesome research assistant Samira Bashiri and posted on Instagram! Yes, our lab is on Instagram @porter_lab_uwindsor! Please, visit, follow and like our photos and videos, it’s another way to be in touch with our lab and research.

Hope you enjoyed the visit!

The Porter Lab was a success at the WCRG2018 this weekend. Our lab marked presence with one of nine Rapid Fire Session presentations and had a total of 10 posters presentations from a total of 58 conference posters. One of our students received a Student Research Excellence Award for his poster presentation and a Porter Lab alumni also received a Student Research Excellence Award for her poster presentation.

Thanks for visiting and please leave comments below.

Tweet

The Porter Lab new logo was designed by our talented Ph.D. student Martin K. Bakht. The logo depicts the main research areas of our lab.

The P of Porter shows the following:

Asymmetric Cell Division

Speedy Cell cycle

The  L of Lab shows the following:

DNA technology

Zebrafish used by our Lab as a xenograft model

The colours match the University of Windsor, Windsor Hospital and WCRG logos, connecting research and health care.

Hope you enjoy our new logo, please leave your comment below.

Elizabeth Fidalgo, Ph.D

Tweet

Tweet

Out with the Old, In with the New!

Biological aging or senescence is the steady decline of cellular function with age. There are a number of rationales that explain why senescence occurs, including changes in gene expression, or damage that is accumulated throughout the lifetime of a cell. Many questions surrounding how to overcome senescence by means of slowing it down, stopping it or potentially reversing it, are the focus of my research project.

The cell cycle lies at the heart of understanding senescence. Teasing apart cell cycle regulation mechanisms and how they are involved in bypassing senescent barriers is critical, especially regarding implications that these mechanisms may have on cellular reprogramming, neural stem cells and tumourigenesis of the central nervous system.

My research focuses on the role of Spy1, a cyclin-like protein, which has been shown to override replicative senescence. As has been previously mentioned by my lab mates, Spy1 can bind, and activate CDK1/2 uniquely without the need of activating phosphorylation and dephosphorylation events to take place. My curiosity with cellular senescence began when I started elucidating Spy1’s role in overcoming reprogramming induced senescence; a unique senescent barrier that somatic cells succumb to on their way to induced pluripotency. Induced pluripotent stem cells have changed the world of regenerative medicine and remain a hot topic with researchers around the globe. We’ve gathered some interesting data that suggests Spy1’s role is essential in overriding reprogramming induced senescence and this is unique to other cyclins. We’ve shown that this leads to an increase in the number of induced pluripotent stem cells that are created from the process. We are currently picking apart some interesting mechanisms of why this may be.

Another piece of my project focuses on Spy1’s role in the neural stem cell population of the brain. We have created a transgenic mouse model system where we can induce the expression of Spy1 under the Nestin+ stem cell population of the brain at any given time, allowing us to investigate its role in neurogenesis and neural stem cell populations throughout life. To date, I’ve been able to show the inability of these cells to terminally differentiate into neurons. Interestingly, although cells expressing Spy1 have increased proliferation rates, their population is maintained throughout life. This naturally brings forward hypotheses of how this stem cell pool is maintained into old age, and what this means to the whole organism in terms of central nervous system tumourigenesis and neurodegenerative diseases. I am lucky to work alongside an amazing research associate in the lab, Dr. Dorota Lubanska, as well as a group of highly motivated and dedicated undergraduate students in a concerted effort to contribute to science every day.

It All Comes Full Circle!

All scientists, whether they are biologists, physicists, chemists, computer scientists, to name a few, are driven to their work because they want to make things better. The knowledge that we gain from science helps us figure out novel ways to fight diseases, create energy, protect natural habitats, connect populations, overcome adversity and much more.

As demanding as a Ph.D. can be, the privilege of getting to be creative and answer important questions in a fascinating environment truly makes up for the workload. Answers to these questions will help us understand the world more and more every day. However, the curiosity behind research questions inevitably means that failure will occur, sometimes more than we’d like. Experiments have to be repeated over and over. Some days it gets hard to remind ourselves of our goals and our vision, especially days when we are fed up with yet another failed experiment. I must note, that with persistence, patience, and continued motivation, you’ll happily surprise yourself by learning something fascinating along the way and ultimately contributing to science.

Induced pluripotent stem cells have taught me the importance of resetting and redefining. Every day we have the potential to re-evaluate our priorities. One thing that keeps me motivated and helps me escape the repetitiveness of the lab bench is communicating my science to others. I truly value how important public education is, and how everyone should be encouraged to gain insight into what I do as a scientist and to support research. This way, I am also challenging others to reset and redefine their views on research. In addition, cellular senescence constantly reminds me that our lives do in fact have an end. It is imperative that we are continually aware of this and adapt the mantra of “sometimes you win, sometimes you learn”. If put into perspective, nothing in science is ever a loss.

Ingrid Qemo, Ph.D. candidate
 

Tweet

Would you ever believe that a fish no bigger than your fingernail could provide information on the best form of therapy for cancer treatments? Well, it’s true! In the Porter lab, the zebrafish group is a part of the Breast Group with our focus being on using fish, specifically zebrafish, to answer questions about which form of therapy is best for individual breast cancer patients. We focus on how drugs work, the pathways inside the cells that they affect, and how the drugs get metabolized within cell culture and our zebrafish model.

Zebrafish are a great model system to use to study heart diseases, neural development and neurotoxins, Alzheimer’s, muscular dystrophy, cancer, and much more! But, do fish have any similarities to humans to study human diseases? Interestingly, yes, they do! They are vertebrates, which are animals with backbones and they share more than 75% of the genome with humans. They have organs similar to humans and have a brain and spinal cord. With an internal environment similar to humans, their responses to cancerous cells and cancer treatments are true to how humans will respond. Zebrafish embryos and larvae are translucent, so we can track the cells throughout the zebrafish daily. We can watch tumours form and regress without any invasive procedures. What’s even greater about using these fish is they have an innate immune system when they are born, but their fully developed immune system doesn’t come into play until 2 weeks after they are born. What this means is we can inject human cells into the fish and they won’t get rejected, but unlike other animal models, the cells are still exposed to some immune system regulations. With all of this, we can now approach new biological questions that I’m so excited to share with you!

Breast Cancer & Treatments

Breast cancer isn’t just one disease. It varies with each patient, which is what makes it difficult to treat at times. We can simplistically separate it into two separate groups or subtypes; hormone receptor positive and triple negative. Each group is controlled by many different factors.

Hormone Receptor Positive: Patients grouped into this subtype in the clinic means their cancer has at least one of three hormone receptors; estrogen, progesterone, and/or human epidermal growth factor 2 (Her2). A benefit of this subtype is these receptors can be targeted with specific drugs to stop the cells from dividing; however, 10-20% of the patient population can develop resistance to these targeted therapies over 10 years and relapse can still occur. A major question is how does resistance or relapse occur? While there are many possible roles, we were able to dissect one mechanism earlier this year.

In February 2017, we published a paper in Oncotarget 2017; 8:23337-52 which described how our protein of interest, Spy1, regulates the estrogen receptor and its pathway changing its response to hormone therapy. As mentioned in previous posts, Spy1 is a protein that promotes increased cell division by binding to CDKs.  I was able to show that when I expressed high amounts of Spy1 in breast cancer cells, the estrogen receptor stopped responding to the hormone therapy Tamoxifen. Using our zebrafish model, we were able to confirm this finding by injecting Spy1 elevated breast cancer cells into the zebrafish followed by treatment with Tamoxifen and watching little to no effect on the tumour. When I inhibited the Spy1-activated pathway (specifically inhibiting the kinase ERK1/2), this showed a significant response to Tamoxifen therapy. Knowing Spy1 levels in patients and targeting Spy1 activated pathways could help us understand what treatments will be more successful.

Triple Negative Breast Cancer: Approximately 10-20% of breast cancers are found to be triple negative. Patients in this subtype do not have high levels or any presence of the estrogen, progesterone, or Her2 receptors. These cancers grow independently of these hormones. Without these receptors, targeted therapies are not useful. These patients are usually treated with chemotherapy. Response to chemotherapy is initially high; however, 34% of triple negative patients have a high recurrence rate in only 2.6 years. One of the reasons for this relapse is the mixed population of cells within the tumour. Therapies normally treat the tumour as one whole product with the hopes that all the cells stop dividing and eventually die. But, the mixed population of cells include a high percentage of special cells called breast cancer stem cells. Breast cancer stem cells have been found to evade treatment, making them a dangerous population of cells. Trying to find new treatment options for triple negative patients that increases overall survival and years of disease free status is one of the most important aspects that we are trying to tease apart.

In collaboration with Windsor Regional Hospital, the Porter lab is a part of two ongoing clinical trials. Since Spy1 has been shown to be elevated in more aggressive and invasive breast cancers (BMC Cancer 2012 12:45), and since elevated levels in hormone positive breast cancers affects treatment, we are interested in determining whether high levels of Spy1 in triple negative patient samples can affect their response to chemotherapy regimens as well as new forms of treatment currently entering clinical trials. Our first clinical trial was to look at the levels of Spy1 in hormone positive and triple negative breast cancer. For this, I received patient tissue in paraffin blocks and created multiple tissue microarrays. These tissue microarrays are then stained for different proteins, which can create a signature for each patient. The goal of this trial is to retroactively look at the patient’s response and levels of Spy1 and see if there is a correlation.

For our portion in the second clinical trial, we will receive fresh patient tissue from the hospital, bring it back to the lab and isolate the cells. One of the first experiments we will perform will be injecting the cells into the zebrafish where we can test out the standard chemotherapy most triple negative patients receive in the clinic as well as some new treatments just entering clinical trials, such as synthetic CDK inhibitors.

CDK inhibitors (CKIs) are naturally found in your body and regulate cell division. They stop your cells from dividing too much or too fast. In cancer, there is a decrease in the amount or levels of these inhibitors, meaning cells have less control in stopping cell divisions from occurring. Pharmaceutical companies have found a way to combat this problem by creating synthetic CKIs and re-introducing them to the body as a form of cancer therapy. Annie, a very hardworking and dedicated 3^rd year undergrad who has received the NSERC Undergraduate Student Research Award for the last 2 years, is currently testing out a number of these CKIs using cell culture work and our zebrafish model on triple negative cell lines. She has already begun to see exciting results and has started combining CKIs with other therapies to test their efficacy. Once patient samples are received from the hospital we can then utilize the protocols she has worked so hard to perfect and we can begin seeing results within one week of testing!

Another aspect of our portion of the clinical trial is to determine what effect breast cancer stem cells may have on treatment. A masters student in the lab, Ellen, is working on multiple tools for our lab to use to determine how much of a population is made up of stem cells. Current work around the world use different kits with labelling techniques, but these have shown to be inconsistent. What Ellen is working on will allow for a more standardized and reliable way to detect stem cell populations. With this new detection method, we can sort out the stem cell population and find new ways to treat these cells to increase response rates in patients. After testing this model in cell lines, we will be able to inject the newly sorted stem cells into the zebrafish model for further treatment testing.

Did you ever wonder how cancer could start in one place, like the breast, and move to another area of the body? Well, cancer cells must go through multiple steps to migrate from one tissue to another and then invade that second tissue. In our group, we have a PhD student, Janice, working on what signals are active in all those steps that makes a cell successfully migrate and invade new tissue. She is trying to figure out whether Spy1 increases the rate of migration and the chances of survival when cells reach the new tissue. She is doing a lot of work in cell culture testing out many different pathways to see which ones are driving these processes. She utilizes the zebrafish model to test out what she sees in cell culture, showing an increase in cell migration when Spy1 levels are increased.  Janice has secured a Mitacs Accelerate Studentship through collaboration with Acenzia here in Windsor based on her work with the zebrafish model. Using the zebrafish, she is testing out the toxic effects chemotherapy drugs can have on the zebrafish and looking at how therapies can alter the immune system. Understanding the immune system regulations and changes is important for understanding the responses we see in the clinic.

Prostate Cancer: Why Movember is Important

Our breast group is always growing, with our newest PhD student Martin, coming to us in 2016 after receiving the Ontario Trillium Scholarship (OTS). Martin has just begun looking at a small subtype of triple negative breast cancer that have the androgen receptor and what role this receptor plays in other subtypes of breast cancers. The androgen receptor, like the others already mentioned, is a hormone receptor and is activated by androgens/testosterone. Martin’s interest in this specific subtype comes from his interest and background in studying Prostate Cancer. Prostate and breast cancer are not that different from each other. Hormonal regulation plays a major role in the development of both cancers and resistance to hormone therapy can happen in both. Prostate cancer is one of the most common cancers found in men; however, research, earlier detection methods, and better treatment options have significantly decreased the death rate by 3.3% per year since 2001. Martin has already completed an abundance of work studying the role Spy1 plays in prostate cancer and if there is any correlation between Spy1 and the androgen receptor. His work is focusing on the subtype of prostate cancer, called neuroendocrine transdifferentiation prostate cancer, which forms after treatment with anti-androgen deprivation therapy and loss of the androgen receptor. He is focusing on Spy1 in 3 main pathways; AKT, β-catenin, and CDK1 activation, to determine if other therapies can be used when androgen dependence is gone. He is also focusing on the role Spy1 plays in the migration and invasion of prostate cells using our zebrafish model.

In our cells and our zebrafish our main goal is to find new and better therapeutics for cancer patients. We understand that each patient is different and should be treated based on their specific cancer and genetics. Our Canadian Institute for Health Research (CIHR) funded work is trying to shed light on what role Spy1 plays in multiple pathways that could lead to patient resistance in breast cancer. We are developing new tools and using a new model system to help us get to answers faster and more reliably.

Research can be enjoyable, but it can also be tough, so we try to follow Dory’s motto while we work and we “just keep swimming!” I hope you enjoyed learning about the second half of the Breast Group! Let us know what you think in the comments below!

Rosa Ferraiuolo, PhD

Tweet

Cancer is a complex disease, and the process towards the discovery of a cure is itself complex. It is a step by step process that builds upon previous discoveries and fitting together the pieces of the puzzle until we get a complete picture. We work at understanding cancer at the most basic level, at understanding how to stop the spread of this disease, and ultimately how we can better treat it and prevent recurrence. While the process can at times seem slow, it is important to remember the gains we have made in recent years working towards eradicating this disease.

Breast cancer mortality rates have decreased by almost half since the late 80s, largely due to early screening and better therapy and treatment options upon discovery of cancer. In the Porter Lab we are hard at work, every day working to understand the basic biology of breast cancer so we can contribute to further success rates in the treatment of this disease.

In our lab, we study a protein called Spy1 or Speedy. Spy1 binds to different binding partners, called CDKs, to help make cells grow and divide. A part of the research ongoing in the Breast Group is on understanding at a basic level how Spy1 speeds up cellular growth and division. By understanding these basic processes, we can better understand what goes wrong when a cell transitions from normal to cancerous and how to better stop it.

We know that Spy1 binding to CDK1 or CDK2 promotes cell division, and at high levels, Spy1 can make cells divide faster. There are times though when your cells need to stop and make sure everything is ok to continue. Cells do this by using checkpoints. You can think of them as the security guards of your cells. Their job is to make sure that everything is good to continue. They’ll spot any problems, like damage to your DNA, and stop the cell in its tracks to fix the problem before the cell is allowed to continue. What happens though, if these security guards miss the problem? This means that damage can be left behind and the damage, or mutation, is passed to the next cell. Over time, mutations accumulate and if left undetected and unrepaired, can allow the cell to grow uncontrollably- a hallmark of cancer. Not only can Spy1 promote cell division, it can also sneak past these security guards in our cells allowing damage to go undetected and accumulate. This creates the perfect storm for cancer to develop.

To understand how Spy1 plays a role in the development of breast cancer, we created mutants of Spy1 that are unable to bind to either CDK2 or another binding partner p27. When put into mammary cells that were then transplanted into mice, normal Spy1 was able to rapidly form tumours, while Spy1 unable to bind to its usual binding partners had delayed tumour growth. This work was published last year in Cell Cycle 2016 15 (1): 128-136 and provided novel insight into how we can potentially target Spy1 as a therapeutic option. One problem remained though- we didn’t know what Spy1 looked like. Think of it like this, if the security guard is trying to catch a suspect for sneaking past their checkpoint, they’ll have a hard time putting out a wanted poster if they don’t know what they look like. A description of what they’ve done may be helpful, but to increase the success of catching the suspect, a picture is best.

Until recently, we’ve only had a hypothesis about what Spy1 looks like when bound to CDKs. A recent collaboration with Dr. Seth Rubin and his team from the University of California, Santa Cruz this year lead to a publication in The EMBO Journal (2017). DOI 10.15252/embj.201796905 which described for the first time what Spy1 looks like and how it activates CDKs uniquely to ultimately lead to enhanced cell division. This exciting data gives us better insight on how to better target Spy1 in the treatment of breast cancer.

Data from our lab and others have shown that levels of Spy1 are elevated in breast cancer and levels are tightly regulated during normal development of the breast (Cancer Research 2008 68 (10): 3591–3600). To truly understand the how and why breast cancer develops, another focus of the breast group is on understanding what role Spy1 plays during normal development of the breast. The breast provides a unique system of study as it is one of the few tissues in the body to continually under different stages of development. There are intense periods of cell division during puberty and early stages of pregnancy, periods when cells stop dividing such as during lactation when cells acquire a different fate to be able to produce milk, and massive regeneration and remodeling of the gland when lactation has ended. Often times, when cells turn from normal to cancerous, they hijack mechanisms used in the normal development and turn it against themselves. Understanding how Spy1 may be regulating normal development will provide great insight into how breast cancer develops and how it can be stopped.

To address this, we developed a mouse model that upregulates Spy1 protein levels in the mammary gland, the MMTV-Spy1 mouse, to determine what effects this may have on the development of the gland, and importantly, will this lead to the development of breast cancer in the mouse?

The development of the gland was intensely analyzed, and while the cells did divide more as would be expected with high levels of Spy1, the gland was otherwise normal with successful completion of each stage of development, and no spontaneous tumours. One factor may contribute to these findings: susceptibility. Just like in people, some mice are more susceptible to the development of certain types of diseases or cancer. The particular strain of mouse we chose to develop this model on is actually quite resistant to the development of breast cancer. Our next question was, even though this particular strain of mouse is resistant to spontaneous mammary tumours, what happens when we challenge it with something known to drive tumour formation? Do the MMTV-Spy1 mice get more tumours? The answer to this question is yes! When challenged with an agent that can cause tumours, the MMTV-Spy1 mice get significantly more tumours which tells us that elevated levels of Spy1 significantly increase susceptibility to tumour formation. We know that Spy1 can bypass checkpoints in the cell that are used to detect DNA damage, and are investigating how Spy1 is capable of doing this and if this is a contributing factor to increased tumour susceptibility. Additionally, we are studying the interactions of Spy1 with other known cancer drivers to determine if Spy1 can enhance or initiate tumour development in cooperation with these drivers.

Since the original MMTV-Spy1 mouse model was generated on a resistant background, we then questioned, what happens when you have elevated levels of Spy1 on a background that is naturally susceptible to mammary tumour formation? We have now generated this mouse on a susceptible model and have exciting data to further delve into the role Spy1 plays in mediating normal mammary development, and what the implications of any alterations in these processes are on the initiation and progression of mammary tumour formation. This work is being followed up on by Iulian, a masters student in the lab who is hard at work dissecting the precise mechanisms Spy1 affects to alter mammary development and potentially predispose the gland to the development of breast cancer.

To gain further insight into the essentially of Spy1 in the development of the mammary gland as well as tumour formation, we are employing CRISPR-Cas9 to knockout Spy1 in mammary cell lines to answer the question, is Spy1 required for normal mammary gland development and initiation of tumourigenesis? Additionally, we can make changes directly to the genome of the cell and insert mutations in Spy1 that will ablate is binding to CDK2 and p27. Amy and Catalin, two dedicated undergraduate students in the lab, are hard at work on these techniques and have already produced exciting results that are shedding light on the basic biology and essentiality of Spy1 in the mammary gland.

Often times when we think of research, we like to think of it as a straight line that leads from point A to point B or a simple 1+1=2 math equation. Ask a question, do an experiment and you get an answer. More often than not though, this is not the case. Many times, we come up with a new hypothesis, test it and need to repeat and re-evaluate our experiments. This was certainly the case with the MMTV-Spy1 mouse. After developing this mouse initially, I carefully analyzed all aspects of the mammary gland and waited and waited for spontaneous mammary tumours- and since it was on a strain resistant to mammary tumours- not a single spontaneous mammary tumour. At this point, it would have been very easy to have been disheartened with the result. Instead, I saw there were 2 forks in the road-one lead to making the model on a susceptible background and one was testing susceptibility on our resistant background. Lack of spontaneous tumours a resistant background actually provided us with more answers and avenues of exploration than we had initially thought. What was even more interesting though, was that in addition to the 2 forks in the road, there was also a detour sign. Often times in research, the most exhilarating results are the most unexpected ones- the detours. These are the results that you don’t always go looking for and never imagined you’d find. Sometimes it’s good to take the detour and enjoy the scenic route because you just never know what you’ll find and learn.

While we set out to study breast cancer using our MMTV-Spy1 mouse, something unexpected was discovered. As the male MMTV-Spy1 mice got older I began to notice that they were very prone to weight gain and something just seemed off. I could have easily dismissed this and just focused my attention on the females as this was the intended area of focus. My curiosity and desire to learn got the better of me though and I decided to look into what I was observing. To my surprise, I discovered that not only were we somehow directing expression of Spy1 to the livers of the male mice, MMTV-Spy1 male mice were developing spontaneous liver tumours! While the mouse strain was resistant to mammary tumours, it turns out, it was susceptible to liver tumours. Some mice without high levels of Spy1 did develop liver tumours as well, but mice with elevated levels of Spy1 developed significantly more liver tumours. This was a completely unexpected finding and has yielded important information about Spy1 in regulating the balance between cellular division and regeneration and other natural mechanisms against injury and inflammation in the liver. This project has been worked on by a dedicated team of undergraduate students over the years who have taken on the challenge with me to learn more about this system. This project is currently in the hands of an extremely hard-working undergraduate student in the lab, John. He has taken on the challenge of dissecting the precise mechanism of how Spy1 may be enhancing the development of liver cancer.

While breast and liver cancer may seem far removed from one another, information gleaned from one system may shed light on what is going on in another system. From both the breast and liver, we know that Spy1 plays a critical role in tumour susceptibility. It may aid in the initial events that turn a cell from normal to cancerous and cooperate with other cancer drivers to enhance this process. As with all tissue types and cancers, the processes may not be identical, but understanding at a fundamental level how Spy1 regulates cellular growth and division will provide novel insight and contribute to our understanding of how Spy1 may initiate and drive tumour formation, and importantly what we can do to stop it in its path. Our research is funded by The Canadian Institutes of Health Research (CIHR) and Windsor Cancer Centre Foundation (Seeds4Hope).

I hope you enjoyed learning a little bit about one portion of the breast group and some of the exciting findings!

Bre-Anne Fifield, Ph.D

Tweet

What makes up a true neural stem cell?

What controls its self-renewal and commitment?

How is the symmetry of division regulated and kept in check?

Can we pinpoint the origin of brain cancer?

Questions about molecular mechanisms behind the regulation of cell fate within the mammalian brain have been fascinating scientists around the world for centuries. The effort to address the essential topics is not only a delightful and captivating everyday struggle of neuroscientists but it’s also a part of a global movement and a world-scale attempt to improve and fight for lives of people touched with neurodegenerative diseases, neurotrauma and brain cancer. In Porter Lab, we have that great privilege to contribute to this global effort. The Brain Group, as the vital organ of our lab, is committed to high-quality research, breakthrough discoveries and new, exciting ideas.

Several of The Brain Group projects are focused on the role of a cyclin-like protein Spy1, aka Speedy-1, SPDYA, RINGO A, in neurogenesis and neural types of cancer. We know that Spy1 activates CDK1 and CDK2 in a unique way and promotes the degradation of the CDK inhibitor, p27^Kip1. Since CDK2 and p27^Kip1, play a regulatory role in many developmental events including neurogenesis and these effectors are aberrantly regulated in several aggressive forms of cancer like glioma, we consider investigating Spy1 function of high importance.

NTA Joint Venture

Our brilliant Ph.D. candidates, Ingrid Qemo and Frank Stringer, are the main investigators of Spy1 role in neurogenesis and expansion of neural stem cells. Based on studies of other neural systems in the lab, like neuroblastoma, we’ve learned that Spy1’s cell cycle-mediated effects are key factors in regulating terminal differentiation in neurons. In addition, examination of the developmental time course of the mammalian brain revealed that significant decline of Spy1 levels coincides with events of neuronal differentiation and apoptosis, whereas the peak of expression overlaps with the phase of increased stem cell proliferation.

Now, a question arises about the importance of Spy1 in maintaining pools of adult neural stem cells and potential consequences of its aberrant regulation.

To address this question we were successfully funded by Canadian Cancer Society, initially with Innovation, and later on, with Innovation to Impact grants which allowed us to generate tools and purchase sophisticated equipment required for detailed and high-quality investigation.

Although one of our dedicated undergraduate students, Dalton Liwanpo, is yet to pinpoint specific neural cell populations abundant in Spy1 protein and mRNA, Ingrid and Frank are already utilizing an inducible transgenic NTA mouse model (Nestin-Spy1-pTRE mouse) to obtain upregulation of Spy1 protein in select, Nestin+, populations of neural stem cells. So far, this system successfully allowed for investigation of the spatiotemporal function of Spy1 in vivo. Ingrid’s expertise in primary cell cultures and Frank’s proficiency in immunoassays on tissue sections, which is currently being passed on to Dalton, came together to establish ample data containing surprising results and exciting phenomena, currently put together in a manuscript to be shortly published as an original article.

Although to date, we have not observed brain tumours in those mice, Spy1, due to its unique functions, constitutes a perfect accessory to ”the crime of brain tumourigenesis”, and this is the main focus of currently ongoing studies and in part a leading objective of another undergraduate project thesis by a diligent student and an Outstanding Scholar, Youshaa El-Abed.

The Glioma Enterprise

In spite of the fact we are yet to discover where the story of Spy1 in brain tumourigenesis starts and ends; whether Spy1 is a factor during initiation or a promising therapeutical target, we did establish its role in glioma maintenance and progression. The funding obtained from Cancer Research Society was crucial in the initial groundbreaking phase of this study. With an amazing support of the co-authors and our collaborators, I was finally able to share six years worth of data in a high impact study, Cancer Cell 25:64-76. We utilized primary cell cultures derived from GBM (Glioblastoma Multiforme) patients and isolated CD133+ cell populations, known to have an extremely high capacity of glioma formation upon orthotopic injection into mice. The depletion of Spy1 levels in those cells caused a significant increase in differentiation markers and enhanced frequency of tumorsphere formation. Further investigation revealed a striking effect of Spy1 depletion on the mode of division of CD133+ cells; we found that decline in Spy1 levels caused a highly significant increase in the rate of asymmetric divisions. We are currently building upon this exciting data, trying to further dissect the heterogeneous cell populations of GBM in respect to its established subtypes. Alex Rodzinka, who is an outstanding and a very bright undergraduate student, awarded with the Brain Tumour Foundation of Canada Research Studentship, is attempting to shed light on the essentiality of Spy1 in the expansion of those populations and targeting Spy1 for their potential eradication, which is tested using Zebrafish platform.

Another burning aspect, awaiting answers, is the mechanism behind our results; although we established WHAT it is, that Spy1 is doing in those GBM cells we still don’t know HOW. Mat Stover is The Brain Group’s only MSc student, his persistence and dedication are exactly what’s needed to take on this task. In addition to molecular pathway determination, Mat is studying how changes in Spy1 levels affect cell cycle profile in glioma subpopulations and how we can utilize that modulation for therapeutic purposes.

The complexity of GBM makes it extremely hard to treat and ironically that complexity expands as our knowledge about it advances, hence, the therapeutic progress always ends up behind.  Can we win this race? Current literature suggests that the patient-tailored treatment has the potential for best clinical outcomes. Now, to determine individual approach, should the patients be evaluated genomically only? Or proteomically, phosphoproteomically, metabolomically or by the cell subpopulation composition? Should we search for additional tools to predict the response of a tumour to therapy?

Tumour Stress Alliance

Our collaborators from Henry Ford Hospital (USA) have developed imaging method (DCE-MRI) module to measure brain tumour physiology and tissue properties such as cellularity, Extracellular space, and perfusion.

Patients presenting with low rates of tumour perfusion due to solid stress (pressure from solid components of a tumour) causing extensive hypoxia and impaired drug delivery, exhibit poorer prognosis with worse chemotherapy response and shorter survival in comparison to patients with high tumour perfusion. Therefore, a tool allowing for measurement of the tumour solid stress and for subsequent therapy response prediction could make a substantial difference.

In our lab, we were able to develop, optimize and validate a 3D platform mimicking patient brain tumour growth in vitro. This system offers an extensive flexibility of environment manipulation and a high throughput approach for drug testing, which are impossible in vivo. Therefore this model has become an in vitro approach to study and compare, to the results obtained through DCE-MRI, the biology of the tumour under solid stress. Jonathan O’Beid is a very enthusiastic undergraduate thesis student who has gained ample of experience on immunostaining of DCE-MRI- assessed brain tumour tissues and analysis of the expression of crucial stress related marker proteins. Jonathan is utilizing the 3D system to manipulate solid stress-related factors and study their effect on drug response in vitro.

Due to the fact that our 3D system allows to entirely control tumour growth and to establish a high throughput time course, we are able to collect data on changes to the cellular layers of our tumour model. The results can be utilized to mathematically model tumour growth before, during and post-therapy and how the arising internal solid stress affects the cellular composition. We are currently in process of collecting and analyzing data which will be forwarded to our collaborators at the Department of Engineering. They are motivated to establish a mathematical approach to the brain tumour growth and drug response prediction.

Our novel 3D platform is fully patient customized and, in combination with the above unconventional interdisciplinary methods, it can, not only, potentially move forward our knowledge on GBM biology but also become a drug response prediction tool, pointing at the necessity of  diverse fields of science to come together in understanding to tackle the most complex phenomena.

Medulloblastoma Incorporated

The medulloblastoma story is slowly coming together as it is one of the projects in our lab which are being passed from hands to hands of very keen and skilled undergraduate students who take over, one after another, bringing all the pieces together at the end. This project was funded by  Brain Tumour Foundation of Canada which also generously offered several Research Studentships related to medulloblastoma studies in our lab.

Over the years we have discovered that medulloblastoma cells cultured as neurospheres enriched in Spy1 but not in Cyclin E1, suggesting a unique role of Spy1 in this particular tumour. Utilizing in vivo zebrafish assays we found that Spy1 levels downregulation is essential in increasing efficacy of medulloblastoma response to synthetic CDK inhibitors.

Philip Habashy, who took over this project, performed further analysis of FACS derived stem-like medulloblastoma cell populations and revealed that Spy1 is an important factor in maintaining their character. Interestingly, Phil’s curiosity made him shift gears recently and work on a novel idea, together with Dr. Huiming Zhang (University of Windsor, Biology), trying to address the role of GABA_B receptor activity in the proliferation of medulloblastoma cells.

The Brain Group’s mission is to dissect mechanisms responsible for control over the fate of neural stem cells and progenitors utilizing diverse in vitro and in vivo systems. The data obtained on Spy1 role in glioma suggest that this unique protein has a potential to become an important therapeutic target. Overall we hope to contribute to better understanding of brain cancer biology and finding new potential ways to target tumours in a specific and efficient way.

I hope you enjoyed getting to know a bit about the Porter’s Lab Brain Group; let us know your thoughts in the comments below!

Dorota Lubanska, Ph.D

Tweet

The research in Porter Lab is divided into 4 main groups, one research associate fellow is responsible for the projects, grants, and students into each group. I’m Elizabeth Fidalgo, Ph.D., and I’m the leader of the Tuberin group. As the group name states our group studies a protein named Tuberin. This protein is a Tumour suppressor protein that controls cell growth and cell proliferation. Mutations in the Tuberin gene (TSC2) lead to several diseases, including Tuberous Sclerosis where patients present with large benign tumours (hamartomas) that affect primarily the skin, heart, brain and kidney and lead to compounding problems like autism and seizures. Mutations in Tuberin also result in Pulmonary Lymphangioleiomyomatosis (LAM), a cyst condition that affects the lungs and surrounding tissues in primarily young women. Mutations in TSC2 have been found in several cancers, including cancers of the skin, breast, kidney, and brain. As you can see, Tuberin is an essential protein for the control of cell growth and proliferation.

My group was the first one to report the role of Tuberin in the regulation of an important checkpoint in the cell cycle. Our results were published in the Cell Cycle 10: 3129-3139 with the title: The tumor suppressor tuberin regulates mitotic onset through the cellular localization of cyclin B1. Since then we have been investigating the mechanisms behind the essential control of cell division by Tuberin.  Understanding the basic biology is necessary for us to understand what this protein does in disease. This work is the foundation of our NSERC (Natural Science Engineering Research Council of Canada) work.

Tuberin is a large protein (200KDa) and is regulated by phosphorylation and it’s the main player of diverse pathways inside a cell.  It isn’t easy to work with it, so my group has developed tools to make a little bit easier to understand the functions of Tuberin towards the cell cycle. One of the first tools we developed using molecular biology techniques is a G2/M reporter, a vector that is transfected into the cell to monitor the Tuberin checkpoint. The cells turn blue when at the checkpoint, this way we can monitor the time that takes for the cell to move from one phase of the cell cycle to other. We’ve published the engineering steps for the construction of this reporter in Cytotechnology 2016 (1): 19-14 Title: Derivation of a novel G2 reporter system. This work was largely conducted by an undergraduate student in our lab, Sabrina Botsford.

Another tool under construction is the BiFC system (Bimolecular Fluorescence Complementation) where the cells turn yellow when Tuberin is actively participating of the checkpoint. These fluorescent tools are important because through them we can use time-lapse fluorescence microscopy and flow cytometry techniques to better understand the functional role of Tuberin in the cell cycle. This BiFC project was started by another undegraduate student, Marisa Market. We are also genetically modifying the genome of HEK293 (kidney) and HeLa (cervical tumour) cells using CRISPR-CAS system. We are introducing TSC2 clinical mutations in the genome of these cells lines to study the pathways affected by these mutations in hopes of revealing how these mutations regulate aberrant cell division. This basic information is essential for research to lead to new treatments for diseases ruled by Tuberin mutations.

This fundamental work supported by NSERC has provided the foundation for several other projects. We collaborate with Dr. James Gauld (Chemistry/Biochemistry UWindsor) on a Seeds4Hope grant sponsored by our local Windsor Cancer Centre Foundation (http://windsorcancerfoundation.org/seeds-4-hope/). This work is figuring out the important binding regions between Tuberin and its partners using computational (computer-based) approaches. In 2013 I was awarded a Seeds4Hope grant to study the role of Tuberin in the formation of Medulloblastoma, the primary brain cancer affecting children; an undergraduate student, Santo Spencer Briguglio, was supported to work on this project by the Brain Tumour Foundation of Canada. We are also currently working with Dr. Andrew Swan (Biology, UWindsor) to expand our cellular studies of Tuberin-Cyclin B1 into an in vivo Drosophila (fly) system. Our results have been presented at many local and international conferences.

One of the strengths of our group is the training of undergraduate and graduate students. We’ve trained several brilliant students, some of them have received awards/fellowships from our department, university, and the province and most have been successfully accepted to Medical and Pharmacology schools through Canada.

Being a tough protein to figure out we often joke that this project trains students to be strong! As an example, one of our past Tuberin undergrads Ryan Ard did his Ph.D. in the Allshire lab in the UK and is now a postdoc in the Marquardt lab in Denmark. Ryan received an international scholarship and has 7 publications including a Nature Communications paper. We are proud of our successful students! We currently have a great group: 2 undergrad thesis students (Jackie Fong and Gillian Denomme), an MSc student (Adam Pillon) and a Ph.D. student in collaboration with Dr. Andrew Swan (Mohammed Bourouh). We are looking for great things to come from this team.

Hope you enjoyed to know a little bit more about one of the research groups in the Porter Lab.

Please leave your comments and/or suggestions below, I’ll be happy to answer them.

Elizabeth

Tweet

Why Research?

The world is constantly changing, evolving and facing new obstacles; climate change, political and social adversity, barriers of equality, poverty, disease, changing population demographics – the list goes on. To the outside world, the workings of Universities are largely unknown. Higher education itself is often viewed as a machine to pump students with information from textbooks – miraculously spitting out doctors, lawyers, leaders, inventors, activists. A professor is a glorified teacher given time to sit and contemplate the problems of the world, an absurd idea.

Within the University system there isn’t much more clarity. Top administrators and leaders in Universities often view the system as a business, bringing in dollars for ‘bums in seats’. For most professors lecturing ‘textbook material’ comprises as little of our time as possible and is not what we value ourselves for; despite this being almost entirely what we are considered to contribute to society. In reality, a successful University doesn’t just train students to perform a specific ‘job’ – Universities encourage independent thought, creativity and innovation. They ‘teach’ grit, determination, ambition, leadership. Universities accomplish this through professors that conduct research and its principles, but how research contributes to these high level outcomes is largely not appreciated.

‘Research’ is an amorphous term that in an abstract way embodies what it should achieve; constant thought, re-evaluation and discussion. Research refers to tackling any unknown by leveraging existing knowledge, methods and analysis. Research objectives and principles, from a scientific perspective, should be simple and organized; but discipline-specific culture, history and politics blurs the path – often so much that even the most level headed can get lost in the fog. It is little wonder why the outside world has a hard time understanding what it is that professors do and why research is important.

Why is research important?

I’ll use my own lab as an example. Our lab studies how cells grow and divide, and how this changes through development and in disease states like cancer. We use complicated techniques that take years to properly train; including making viruses and using novel methods to alter the DNA of a cell, growing human cells in new ways that mimic what they do in the human body and using animal models to dissect what causes diseases in humans and new potential therapies. Research today, just as everything in Science, is more demanding than it was even a decade ago. Students have thousands of papers at their fingertips, and mountains of data readily available and constantly growing, and they are expected to absorb them all and come up with the next logical step for a field that they are just learning about. Students have to learn the latest most cutting edge technologies, and some of the most pressing questions may require that you advance this technology yourself. Students, like professors, are faced with a system that doesn’t discuss, or seem to understand, the value of research – yet they sign up. They come into a lab like mine and become completely immersed. WHY – because they love to figure out why cells do what they do. They want to understand diseases like cancer. They want to understand why and how we develop. They are curious, hardworking, intelligent and driven – and we are providing an environment where they can let this all loose. Simple.

What happens after that is where the magic lies. Students come into my lab and fail, sometimes miserably. I take the brightest, fastest-thinking individuals, put them on the steepest learning curve imaginable and tell them that to get one small answer they have to set up numerous tedious, repetitive experiments and then repeat this cycle multiple times. After each experiment they have to scrub their own dishes, ensure everything is back in the proper spot and ordering complete – oh and don’t forget to put your garbage in the correctly labeled bins. Inevitably experiments fail and fail again until the students troubleshoot a solution. Some students feel overwhelmed, exhausted, disillusioned, some pissed off. Research associates and I struggle to deal with these students; each with their own individual personalities and problems – we train and provide a framework, hold some hands, let some go under water – but amazingly, together, some students swim – some beautifully. Bright minds come up with new ideas that excite us as a group – and slowly we begin to get answers to our questions.

Some answers will lead to new therapies for cancer, some will make sense of problems occurring during development, and others will go into textbooks to be taught in the classroom to our students so they can make the next steps forward. Each question that we are actively tackling is hope for patients dealing with devastating diseases. Each tedious step forward makes a bit more sense of our world.

Beyond the puzzles that we put together, and the knowledge that we contribute, are the people that it took to get there – thinking, repeating, working, struggling and digging deep. Learning isn’t just about absorbing the theory behind what we do – it’s figuring out ‘why’ you are doing it and ‘how’ to get to the next step. Some cannot handle that answers come slowly, that the path is not clear and destination undefined; others thrive on it. But one thing is certain – research ‘teaches’ what no textbook can – it teaches tenacity, grit, determination and all while encouraging creativity and inspiring teamwork and leadership.

Lisa Porter

***************************

I’m very proud of the number of ‘swimmers’ that have emerged from the Porter lab. I am lucky enough to have 4 bright, motivated and dedicated group leaders who help push and define who we are as a group. My lab provides me with a constant source of energy, ideas and inspiration.

In this blog I’m challenging everyone in my group to provide a short blurb about one of their research questions or papers – and to follow it with one lesson that they have learned along the way.

Thanks for reading and your interest!  Feel free to leave comments, questions, suggestions!

Tweet