A New Role for Folliculin in Cancer Prevention

We are excited to be sharing with you a recently published paper by Woodford et al., exploring the reason why the loss of folliculin (FLCN), the gene mutated in Birt-Hogg-Dubé syndrome (BHD), drives tumour development.

Cancer cells have characteristics that allow them to grow and replicate uncontrollably. One of these is the deregulation of cellular metabolism, in other words, cancer cells can change how they get their energy. To produce energy cells normally break up glucose to produce pyruvate which then interacts with oxygen and releases carbon dioxide (CO2). If there is limited or no oxygen pyruvate is converted to lactate instead. However, cancer cells will rapidly take up glucose and convert pyruvate into lactate, even in the presence of oxygen, which is beneficial for the cancer cell. This shift in cellular metabolism is known as the Warburg effect and is driven by Lactate Dehydrogenase A (LDHA), the enzyme which converts pyruvate into lactate. Interestingly, loss of FLCN has previously been shown to increase LDHA activity. Woodford et al., examined this relationship further and investigated whether LDHA could be targeted to treat Kidney cancer.

Several different experiments were undertaken to characterise the relationship between FLCN and LDHA. Detailed examination of the FLCN protein in a kidney cell line showed that FLCN interacted with 114 different proteins including LDHA and that high FLCN expression reduced LDHA activity. To determine how FLCN reduced LDHA activity the relationship between LDHA and its cofactor (a small molecule needed for LDHA activity) was examined. High levels of FLCN reduced the binding between LDHA and its cofactor likely through alteration of LDHA’s structure thereby reducing the activity of LDHA. Taken together these results demonstrate a new role for folliculin as a binding partner and inhibitor of LDHA.  

Next Woodford et al., examined the significance of this finding in cancer cells. 13 cell lines, including a kidney cell line, showed a metabolic shift and hyperactivity of LDHA. 11 of these cell lines also showed a dissociation between FLCN and LDHA suggesting that the inhibitory effects of FLCN are lost in many cancer cell lines.

The creation of targeted therapies against LDHA has always been an appealing area of investigation for cancer treatments, however creating a target that is specific to LDHA has been challenging because there are other similar proteins. The discovery of FLCN as an inhibitor of LDHA creates a new opportunity for such therapies. Firstly, Woodford et al, showed that FLCN binds specifically to LDHA and none of its similar proteins. They then identified the specific region of the FLCN protein which binds to LDHA.  They produced a series of peptides (a chain of amino acids which is the building blocks of proteins) derived from FLCN to target LDHA based on their findings of how FLCN binds to LDHA. These peptides were tested in normal kidney cells in vitro and those that were taken up by the cells and reduced LDHA activity were selected for further tests. They then examined the response to the peptides in a kidney cancer cell line. One of the peptides named FLCN-10 was able to bind to LHDA and this resulted in cancer cell death. Next tissue samples from a kidney cancer of a BHD patient were treated with FLCN-10. FLCN effectively reduced LDHA activity in the kidney cancer cells.  

Altogether Woodford et al., have demonstrated a new role of FLCN as an inhibitor of LDHA. It binds to LDHA and regulates its activity. Therefore, in BHD, loss of FLCN results in LDHA hyperactivity resulting in the Warburg effect which is beneficial for cancer cells.  An understanding of this interaction between FLCN and LDHA meant Woodford et al., were able to create a folliculin derived peptide which bound to LDHA and inhibited its activity. This discovery could pave the way for a new LDHA targeted cancer treatment not only for BHD patients but other cancer patients as well.

We are sorry that the paper is currently not freely available. Please message us if you have any further questions at contact@bhdsyndrome.org.

References

1. Woodford MR, Baker-Williams AJ, Sager RA , Backe SJ , Blanden AR, Hashmi F, et al. The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect. Nat Struct Mol Biol [Internet]. 2021 Aug 1 [cited 2021 Sep 3];28(8):662–70. Available from: https://pubmed.ncbi.nlm.nih.gov/34381247/

Is Birt-Hogg-Dubé Syndrome linked with colon cancer?

Is Birt-Hogg-Dubé Syndrome (BHD) linked with colon cancer? This is a question we often receive at the BHD Foundation and does not have a simple answer. Colon cancer is one of the most common cancers in the western world and is thought to be largely linked to environmental factors such as diet and age. However specific genetic mutations increase the risk of developing colon cancer and it is yet to be proven whether folliculin (the gene mutated in BHD) is one of them. There are reports of BHD patients developing colon cancer, but current research is not conclusive on whether these cases are linked to BHD or it is just a coincidence. Investigating this link is important as it will help to inform colon cancer screening in the BHD community.

A recent paper by Sattler et al., analysed the frequency of colon cancer in 256 BHD patients compared to 519 non BHD patients(1). We spoke to the lead researcher and BHD expert Professor Steinlein about this paper and delved into why researchers may be getting different results and what needs to be done to provide further evidence on the link between colon cancer and BHD.

What inspired your research looking into the risk of colon cancer in BHD?

Elke Sattler, Dermatology, and I (Human Genetics) started Germany’s only interdisciplinary BHD outpatient clinic more than 10 years ago and, together with Zulfiya Syunyaeva (Pulmonology) we have by now collected nearly 100 mostly multiplex families. Our patients, finding information about possible associations between colorectal cancer and BHD on Google, are often asking about their risk for this tumor type. Furthermore, we always perform extensive pedigree analysis in our BHD families and observed a higher than expected rate of early onset colon cancer. Some months ago we therefore decided to systematically analyse our data.

What did you discover?

We first looked at the frequency of colorectal cancer and found it to be significantly more frequent in BHD patients than in controls. However, the difference between both groups was small, meaning we found only a moderate increase in risk. Such small differences are statistically not very robust and not all previously published studies reported the same effect. Much more important is our observation that in BHD families in which colorectal cancer occurred, it often occurred either at an unusually early age (i. e. before the age 50 years) or affected several family members. This is a pattern well known from HNPCC (hereditary non-polyposis colon cancer or Lynch syndrome), the most common hereditary colorectal cancer syndrome.

There is an ongoing discussion about whether there is a link between colon cancer and BHD and whether BHD patients should get screening. What are the challenges faced by researchers to answer these questions and how can we overcome them? 

The biggest challenge will be the availability of a large number of clinically well characterized BHD families. During the last decade several of such samples have been collected by different research groups but sample sizes still need to be increased. There is also the possibility, that cancer risks differ between ethnic or regional backgrounds. It therefore most likely will take several more years to collect enough large BHD samples from different populations before the BHD research community will be able to give a final answer to the question about a possible association between colorectal cancer and BHD.

What message would you give to BHD patients who are concerned about developing colon cancer?

We are counselling our patients that after age 50 years the colon cancer risk is likely to be comparable to the age-related risk for this cancer type in the general population. However, there might be a small but significant risk for early onset colorectal cancer. We are therefore suggesting to start coloscopy at age 40 (or ten years before the first family member developed colorectal cancer in patients where there is a family history of colon cancer).

Are you working on any other BHD research projects currently and can you tell us a bit about them?

BHD is our main research focus and we are steadily increasing our sample of families. Most of our current research projects are focused on clinical aspects, especially possible associations between BHD and different cancer types but we are also in the planning stage for functional studies.

Altogether this study suggests that although there is not a significant increase in colon cancer, BHD patients may be at increased risk of developing colon cancer at a younger age. Because of this discovery Professor Steinlein advises caution and suggests that BHD patients start colon cancer screening from the age of 40 (10 years earlier than screening for the general population).

It must be noted that due to the variation in results investigating the link between colon cancer and BHD, recommendations may vary in different areas. We recommend discussing any queries regarding colon screening with your BHD doctor. If you would like assistance identifying a BHD specialist please contact us at contact@BHDsyndrome.org or search our interactive map.

The BHD Foundation sincerely thanks Professor Steinlein for discussing her paper with us and the BHD community. The research paper is not currently freely available. Please contact us at contact@bhdsyndrome.org if you have any questions regarding this research.  

References

1.          Sattler EC, Syunyaeva Z, Reithmair M, Dempke W, Steinlein OK. Colorectal cancer risk in families with Birt-Hogg-Dubé syndrome increased. Eur J Cancer. 2021 Jul 1;151:168–74.

Register Now for the BHD Symposium Patient Sessions!

The BHD Foundation is excited to announce that the tickets for the BHD symposium patient-focused sessions are now available. Tickets are free and are aimed at BHD patients and their families and friends.  

There will be two unique sessions exploring the future of BHD.  Please register for each event separately and note you are welcome to attend both sessions or just one (they both work well as standalone sessions).  

Thursday 21st October 7:00pm – 8:10pm (British Summer Time)

The Future of BHD Session 1 

In this session, you will have the opportunity to explore the exciting and innovative research being done into BHD and what this means for the future. BHD experts from around the world will be sharing their work and answering your questions, so together we can look towards a future of curing BHD.  

Register for The Future of BHD session 1 here.

Friday 22nd October 7:00pm – 8:10pm (British Summer Time)

The Future of BHD Session 2

On day two we will have a panel of BHD patients who will be sharing their personal BHD stories and discussing the challenges BHD patients face and how to support those who are newly diagnosed. You will also have the opportunity to ask the Team at the BHD Foundation about the work we do and how together we can raise awareness of BHD.

Register for The Future of BHD session 2 here.

If you have any questions about the event or would like to volunteer to be part of the patient panel please email us at contact@bhdsyndrome.org.

We are looking forwards to seeing you all there!

Extracardiac Rhabdomyomas in BHD

Rhabdomyomas are rare cancers formed from striated muscle. Although they are usually benign, meaning they do not spread elsewhere in the body, they can negatively impact the structures around them so must be monitored. There are two main types of rhabdomyoma, cardiac (found in the heart) and extracardiac (found elsewhere in the body), which is further split into three subtypes: adult, fetal and genital. Although rare, there have been a few cases of cardiac and adult rhabdomyoma reported in patients with Birt-Hogg-Dubé syndrome (BHD), however, there is not enough evidence to determine whether these are incidental findings or are characteristic of BHD. A recent research letter by Bajwa et al., discusses a patient with BHD who was found to have multiple extracardiac rhabdomyomas and explores whether they could be linked (1).

The letter describes a 63-year-old man, with known BHD, who presented with ‘fullness’ under the chin. He had an ultrasound and CT of his head and neck which showed multiple soft masses in his neck. A biopsy was taken whereby multiple extracardiac rhabdomyomas were found. The DNA of the tumours was sequenced and demonstrated loss of heterozygosity for the folliculin gene. This is what happens in BHD-related kidney cancer, where the second copy of folliculin acquires a mutation or is lost, which leads to the formation of tumours. This is not the case in the fibrofolliculomas or lung cysts seen in BHD where mutation in a single copy of folliculin is sufficient.

Bajwa et al., explored the literature and this case to further understand whether there may be a link between BHD and rhabdomyomas. Firstly, it must be noted that it is rare to get multiple extracardiac rhabdomyomas, with only 33 cases having been reported in the literature; this suggests a genetic cause. Folliculin was shown to be mutated in the case of the 63-year-old and although BHD was not diagnosed in any of the other cases, one patient was said to have undefined skin papules, which were not investigated further (2). This suggests it could be the result of BHD.

In addition to multiple cases of rhabdomyoma being recorded in the literature, one study  using a rat model of BHD showed that 12% of rats  developed rhabdomyomas (3).

At the BHD foundation we were really interested by this case report which appears to be the first piece of evidence demonstrating that loss of heterozygosity of folliculin can drive the growth of rhabdomyomas. This case study, and the limited other reports of rhabdomyomas in BHD, provides a stronger link between rhabdomyomas and BHD and warrants further investigation. A database or patient registry which collects information regarding BHD and infrequent manifestations would further consolidate these associations.

References

1.          Bajwa DS, Cook S, Winn R, Winship IM, McQueen A, Husain A, et al. Multifocal extracardiac rhabdomyomas: Extending the phenotype of Birt‐Hogg‐Dubé syndrome. Br J Dermatol. 2021 May 28;

2.          Khalaf MG, Haddad R, Akiki M, Khazen J, Melkane AE. Multifocal adult rhabdomyoma of the head and neck: case report and systematic review of the literature. Vol. 50, International Journal of Oral and Maxillofacial Surgery. Churchill Livingstone; 2021. p. 327–34.

3.          Bondavalli D, White SM, Steer A, Pflaumer A, Winship I. Is cardiac rhabdomyoma a feature of Birt Hogg Dubé syndrome? Am J Med Genet Part A [Internet]. 2015 Apr 1 [cited 2021 Jul 8];167(4):802–4. Available from: https://pubmed.ncbi.nlm.nih.gov/25655561/

Cassandra’s BHD Story: Pneumothorax

Today is World Pneumothorax Day. Familial pneumothorax accounts for around 10% of all spontaneous pneumothoraces. Of those that can be diagnosed with a genetic cause, BHD is the most common diagnosis but is often not thought about in the clinic. The BHD Foundation would like to change this and raise awareness of BHD and pneumothorax.

We also spoke to Cassandra a member of the BHD community about her BHD diagnosis and pneumothoraces. Cassandra explained how recurrent pneumothoraces resulted in surgery and key things to consider in the recovery process. She also discussed how she would approach discussing BHD with her daughter and the importance of information. You can watch her interview below. A transcript is also available here.  

Worldwide LAM Awareness Month

June is Worldwide LAM Awareness Month which aims to raise awareness and educate people about Lymphangioleiomyomatosis (LAM). LAM is a rare disease caused by mutations in the tuberous sclerosis genes. It affects the lungs, kidneys and lymphatic system and almost exclusively affects women. Recognising the symptoms of LAM is important in order for patients to access the treatment they need early. The most common presenting symptom of LAM is worsening shortness of breath and a history of recurrent pneumothoraces. Additionally, patients can experience bloating due to fluid around their lungs and stomach (from the disruption of their lymphatic system) and angiomyolipomas, which are a type of benign tumour that can cause pain and bleeding. Although there is no cure for LAM, the progression of the disease can be slowed by a drug called Sirolimus (Rapamycin).  

Due to its similarities with BHD, researchers often study both conditions. We talked to Professor Elizabeth (Lisa) Henske, Director of the Center for LAM Research and Clinical Care, Brigham and Women’s Hospital, about her work in LAM and how this translates to the BHD community. Her lab’s discovery of Tuberous Sclerosis Complex 2 (TSC2) resulted in the first FDA approved drug for LAM.

Could you tell me about your discovery of the TSC2 mutation and what that meant for patients? 

LAM occurs in two forms:  in women with tuberous sclerosis complex (TSC) and in women who do not have TSC (referred to as sporadic LAM).  The Henske Lab discovered that sporadic LAM is caused by mutations in the TSC2 gene.  A few years later, it was discovered that the TSC proteins inhibit the mTOR pathway (a pathway involved in cell growth), and Dr. Vera Krymskaya demonstrated that the mTOR pathway is hyperactive in LAM cells.   Together, these discoveries paved the way for a pivotal clinical trial led by Dr. Frank McCormack, which was published in the New England Journal of Medicine in 2011.  This trial proved that treatment with sirolimus (Rapamycin), an oral inhibitor of the mTOR pathway, stabilizes lung function in women with LAM.  In my own clinical practice, I can see the tremendous benefit of sirolimus in so many women with LAM, allowing them to work, care for their families, travel, exercise, and live a life that is close to “normal.” 

What research is currently being done into LAM? 

Some of our most urgent research questions include:

1) why does LAM affect almost exclusively women?
2) can we use treatments that block estrogen’s actions to treat LAM? 
3) how does the immune system promote the growth of LAM cells?
4) why do LAM cells cause cystic destruction of the lungs? 

We are excited to be working with Dr. Wei Shi, a leading developmental biologist at Los Angeles Children’s Hospital, to understand how both LAM and BHD lead to cystic lung disease. 

What difficulties have you come across in researching LAM? 

With any rare disease, access to tissue samples (for example, lung and kidney samples) is absolutely essential to progress.  We have been very fortunate that The LAM Foundation (based on Cincinnati OH) has helped make samples available for research.  We are also lucky to have an exceptionally collaborative and connected LAM research community, including many dedicated scientists and physicians who are committed to curing LAM.  Dr. Joel Moss, who leads the LAM program at the National Institutes of Health, has had an especially positive impact on these collaborative efforts.

From your experience what are the main differences/similarities between the symptoms of BHD and LAM? 

There are important similarities and also important differences between BHD and LAM.  The similarities include the risk of lung collapse (pneumothorax) and the risk of kidney tumors (renal cell carcinoma in BHD and angiomyolipomas in LAM).  One important difference is that in BHD, it is unusual for shortness of breath to develop.  In LAM, many women develop shortness of breath, and some become dependent on oxygen therapy, especially if the LAM is not treated early in the disease.  Another difference is that LAM affects almost exclusively women, while cystic lung disease in BHD occurs in both men and women.

What can the BHD community learn from the research into LAM? 

LAM research has brought together a highly collaborative group of clinicians and scientists.  The scientists are diverse in their background and approaches, including biochemists, developmental biologists, cancer biologists, cell biologists, geneticists, and more.  This allows LAM to be studied from many different angles.  Regular scientific and clinical meetings allowing new data to be discussed and ideas to be shared have propelled LAM research breakthroughs. 

What can we do to raise awareness about rare conditions such as LAM and BHD?

One of the best ways to raise awareness is to make clinically relevant discoveries.  In LAM and TSC, breakthroughs have consistently boosted awareness among both scientists and clinicians.  This has a “positive feedback” effect, with more awareness leading to even more progress. 

The BHD Foundation sincerely thanks Professor Elizabeth (Lisa) Henske for taking part in this interview and sharing her insights on LAM and BHD. If you a researcher working on BHD we would love to hear about your research. Please contact us at contacts@bhdfoundation.org

To find out how you can get involved with LAM awareness month visit the LAM Foundations webpage. The LAM Foundation is a wonderful resource for both clinicians and members of the LAM community and they have several useful resources for raising awareness about LAM. Their centres of clinical excellence also support BHD patients.

World Kidney Cancer Day

Today is World Kidney Cancer day and the theme is ‘We need to talk about how we are feeling’. A patient survey revealed that 96% of kidney cancer patients experience psychosocial problem but less than half talk about it. It is not always easy to discuss how you are feeling but research shows that it does improve wellbeing.  World kidney day is striving to make this easier.  They have produced a personalised psychosocial wellbeing report for you to complete with recommendations on how to improve your mental wellbeing. The report is available here.

The BHD Foundation is talking about kidney cancer and wellbeing because up to 30% of patients with BHD develop kidney cancer and we want you to know you are not alone. In addition to initiatives such a world cancer day there is a welcoming patient lead BHD Facebook group who are always happy to answer questions and share their stories. Earlier this year we also interviewed a member of the BHD community about her experience being diagnosed with kidney cancer and it is available to read here. The BHD Foundation can help connect you with others who have been diagnosed with BHD.

I ♥ Small Charities Day

The BHD Foundation is taking part in #SmallCharityWeek. The aim of this week is to raise awareness of the UK small charity sector making a difference across the world. As a small charity, the BHD Foundation (run by the Myrovlytis Trust) is getting involved in this event and we would love the BHD community to get involved too!  
 
Monday 14th June is “I ♥ Small Charities Day”. The team at the BHD foundation will be posting a picture of themselves on Twitter and Facebook holding a poster saying how much we love our charity and why. We think this is a great way to connect with the BHD community so you get to see some of our faces behind the scenes! We would also really love to see some of you too – we would be nothing without the strong community we have.  
 
We have provided a link to a downloadable poster for you to adapt and we would love to see them across social media using the hashtags #ILoveSmallCharities  #SmallCharityWeek #BHDsyndrome #SCWeek2021. 

Alternatively, you can share the poster without a photo on social media with the same hashtags – or even just share your feelings in a FB post or tweet!  

We will be gathering all the responses and putting them together.
 
The poster is available here!


 

Loss of folliculin causes cellular dysfunction of pleural mesothelial cells

People with Birt-Hogg-Dubé syndrome (BHD) have a 25% chance of developing at least one spontaneous pneumothorax (collapsed lung) in their lifetime and many people will have several. A pneumothorax occurs when air enters the lining of the lung (known as the pleural cavity) causing the lung to be compressed. The size of the pneumothorax usually determines the clinical presentation and required intervention ranging from a conservative watch and wait approach to surgery. Pneumothoraces in BHD patients are primarily thought to be caused by the lung cysts (which are seen in 80% of BHD patients) bursting. However, it has been suggested that this is not the only cause (1).  Okamoto et al., conducted the first study assessing the effect of folliculin haploinsufficiency (one mutated folliculin gene) on pleural mesothelial cells (PMCs) (2). PMCs are a single layer of cells that blanket the lung and chest wall acting as a protective barrier. If this layer is disrupted it can result in a pneumothorax.  Studying these cells increases our understanding of how BHD causes pneumothoraces and potential drug targets to prevent them.    

Okamoto et al., collected PMCs from 32 patients who developed spontaneous pneumothoraces: 12 BHD patients and 20 other patients who had no underlying lung conditions (control group). The purpose was to compare the cells in both cohorts to see if there were any differences. The cells were examined under a microscope and they found that PMCs in BHD patients were cuboidal in shape compared with the cobblestone appearance of the control group.

Next, Okamoto et al., looked at the expression of genes involved in cell adhesion (binding of cells together). The expression of E-cadherin, which is involved in cell adhesion was reduced in BHD PMCs. To investigate the effects of reduced E-cadherin on BHD PMCs they assessed cell adhesion in Petri dishes. A significantly greater number of cells detached in the BHD PMCs compared to the control and then underwent anoikis (programmed cell death due to loss of attachment to other cells). In addition to reduced cell adhesion and anoikis, Okamoto et al., demonstrated delayed cell growth and reduced migration in BHD PMCs compared with the control group.  PMCs are involved in maintaining the health of the lining of the lung and repairing any disruption, so need to be able to move and grow. Altogether this suggests that folliculin haploinsufficiency causes PMCs to be disrupted through loss of adhesion and cell death and the repair process hindered resulting in pneumothoraces.  Okamoto et al., suggest these changes to PMCs function may be due to reduced activation of the E-Cadherin-LKb1- AMPK pathway which is a known pathway affected by folliculin loss (3).

Altogether Okamoto et al, found that folliculin haploinsufficiency causes changes in the shape of PMCs in BHD patients and impairs the function of the cells, potentially through the E-Cadherin-LKb1- AMPK pathway. This is important as the disruption of PMCs may be a cause of pneumothoraces in BHD. Further research is required to determine how exactly PMC disruption causes pneumothoraces and whether the pathways affected could be targeted to prevent them.

References

1.            Ohata M, Suzuki H. Pathogenesis of spontaneous pneumothorax. With special reference to the ultrastructure of emphysematous bullae. Chest [Internet]. 1980 Jun 1 [cited 2021 Jun 8];77(6):771–6. Available from: http://journal.chestnet.org/article/S0012369215456168/fulltext

2.            Okamoto S, Ebana H, Kurihara M, Mitani K, Kobayashi E, Hayashi T, et al. Folliculin haploinsufficiency causes cellular dysfunction of pleural mesothelial cells. Sci Rep [Internet]. 2021 Dec 24 [cited 2021 Jun 8];11(1):10814. Available from: http://www.nature.com/articles/s41598-021-90184-9

3.            Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, et al. Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-Cadherin, LKB1, and AMPK. Cell Rep [Internet]. 2014 Apr 24 [cited 2021 Jun 8];7(2):412–23. Available from: https://pubmed.ncbi.nlm.nih.gov/24726356/