The PI3K/mTOR inhibitor GSK2126458 is effective for treating TSC solid renal tumours

Tuberous sclerosis (TSC) is an inherited tumour syndrome that shares clinical similarities with Birt-Hogg-Dube Syndrome. It is caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR, affecting multiple organs, including the kidney and lung. In the kidney, lesions such as multiple renal cysts and renal cell carcinoma (RCC) can occur. Tumour reduction in TSC patients after treatment with rapamycin, an inhibitor of mTOR, is partial and reversible probably due to feedback activation of Akt. In their new study, Narov et al. (2017) test the efficacy of GSK2126458, an inhibitor of PI3K/mTOR, in comparison to rapamycin, for treatment of renal tumours in genetically engineered Tsc2+/- mice, that spontaneously develop various lesions in the kidneys. Both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Both GSK2126458 and rapamycin suppressed proliferation of tumour cells. However, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are needed to test whether rapamycin in combination with GSK2126458 can improve anti-tumour therapy.

The kidney lesions of adult Tsc2+/- mice had aberrant activation of the mTOR complex 1 (mTORC1) and mTORC2. The MAPK pathway was also activated in these lesions. To test the anti-tumour efficacy of GSK2126458, the authors first determined the maximum tolerated dose (MTD) of GSK2126458 in Tsc2+/- mice. After this, adult mice were treated for two months with vehicle, the MTD dose of GSK2126458 or rapamycin. Both GSK2126458 and rapamycin significantly reduced total number, size and cellular area of solid renal tumours and other lesion types. Rapamycin caused greater reduction than GSK2126458 in number, size and cellular area of all types of lesions but the difference in reduction of solid tumour burden was not significant. IHC of kidney sections was used to investigate PI3K/Akt/mTOR signalling. GSK2126458 reduced phosphorylation of Akt at T308 but rapamycin did not. It is not known whether this reduction in Akt phosphorylation contributes to antitumour efficacy. The phosphorylation of a part of the Erk1/2 signalling pathway, was reduced in GSK2126458 treated solid renal tumours. The Erk1/2 signalling was inhibited by rapamycin in solid renal tumours. In contrast, reduced phosphorylation of mTOR at S2481, an indicator of mTORC2 activation, was detected in rapamycin treated solid tumours. Ki67 staining was used to assess proliferation of renal tumour cells on treated mice. Both GSK2126458 and rapamycin markedly reduced the median percentage of Ki67-positive cells. Rapamycin inhibited proliferation of tumour cells to a greater extent than GSK2126458. Active caspase 3 was used to test whether treatment induced apoptosis in tumour cells. Interestingly, GSK2126458 significantly increased the median total area of active caspase 3-positive tumour cells but rapamycin did not. Similar results were observed when other apoptosis marker was analysed. To investigate the mechanism behind the increased apoptosis associated with GSK2126458 treatment, expression of p53 and phosphorylation of MDM2 at S166 were analysed by IHC. Renal lesions had a lower level of p53 and decreased phosphorylation of MDM2 in rapamycin treated mice, but not GSK2126458 treated mice, compared to vehicle treated mice. Similar results were observed by Western analysis.

In conclusion, the authors demonstrated that GSK2126458 was effective for treating solid renal tumours. A Phase I clinical trial has recently reported that GSK2126458 is well tolerated in patients treated for multiple solid malignancy types and tumour responses and disease stabilization were observed (Munster et al., 2016). The authors found that GSK2126458 inhibited both mTORC1 and mTORC2 in all types of renal lesions in Tsc2+/- mice but the inhibitory effect of GSK2126458 on mTORC1 was weaker than that of rapamycin. Both GSK2126458 and rapamycin reduced proliferation of tumour cells. However, GSK2126458 increased apoptosis of solid tumours but rapamycin did not.

The clinical similarities between BHD and TSC suggest that FLCN and TSC proteins may function within a common pathway. It may be worthwhile investigating whether combination of GSK2126458 with rapamycin could improve anti-tumour therapy through increased tumour cell death in different disease models.

  • Narov, K., Yang, J., Samsel, P., Jones, A., Sampson, J., & Hong Shen, M. (2017). The dual PI3K/mTOR inhibitor GSK2126458 is effective for treating solid renal tumours in Tsc2+/- mice through suppression of cell proliferation and induction of apoptosis Oncotarget DOI: 10.18632/oncotarget.17215

Characterization of a FLCN mutation associated with RCC

Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé (BHD) syndrome, a rare disease characterized by renal cell carcinoma (RCC), pneumothorax and fibrofolliculomas. In their new study, Bartram et al. (2017) identify a heterozygous mutation in the FLCN gene in a patient with RCC. DNA from tumour and a metastasis was analysed and the authors demonstrated skipping of exon 11 as the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. The FLCN protein was still expressed but it was strongly destabilized and had a different subcellular localization. Both altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation.

In this study, a 55-year-old patient presented with weight loss, bilateral kidney cysts and tumours. He and family members had a history of recurrent pneumothorax. Histology after kidney tumour nephrectomy showed elements of a chromophobe RCC (chRCC) and a small cell carcinoma component. In addition, CT scan showed liver and spleen enlargement, and several lung cysts and pulmonary nodules. Open surgery revealed peritoneal metastases.

Histology of one metastasis showed features of the small cellular tumour component, suggesting that these cells might contribute to the aggressive tumour phenotype in the patient. After surgery, hemodialysis was initiated due to renal failure. Unfortunately, the patient died shortly afterwards as a consequence of the advanced stage of the metastatic tumour disease.

The co-occurrence of chromophobe RCC with familial recurrent pneumothorax lead to the suspicion of BHD syndrome.

BHD-syndrome associated RCC normally show a benign nature and rarely metastasize. Here, the patient suffered from metastases and pulmonary lesions. The metastases did not show the classical characteristics of the chRCC but rather a small-cell morphology. Since the chRCC showed different levels of dedifferentiation towards the areas of the small cell tumour component the authors speculate that the small cell carcinoma arose from the chRCC by acquiring further genetic alterations.

FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. In silico analyses by bioinformatic tools predicted this variant to be likely pathogenic. FLCN MLPA analyses were consistent with deletion of the second FLCN allele in both tumour tissues.

The metastasis appears to be linked to BHD since it showed loss-of-heterozygosity in the FLCN gene. It will be interesting to see in future cases whether this entity is associated with BHD syndrome.

To validate the mutation’s impact on splicing of the FLCN transcript the authors generated minigene constructs containing either the FLCN  wild-type (WT) or the mutant sequence. These minigene constructs showed that the deletion indeed abrogated the acceptor splice site of exon 11, leading to skipping of exon 11 and fusion of exon 10 to exon 12 which generates a frameshift and premature stop codon.

To investigate whether the predicted FLCN protein is expressed the authors analysed protein expression in HEK293T cells. Overexpression of a FLAG-tagged cDNA revealed that the mutant protein is expressed, however, at lower levels than the WT protein. A commercially available anti-FLCN antibody detected the overexpressed mutant protein in Western Blots and this antibody was used to analyse the endogenous expression in patient tumour tissue with a clear signal being obtained by immunohistochemistry. To confirm this the authors generated transgenic cell lines using the TALEN technology that expresses GFP-fused versions of either the WT or the mutant protein and mimic the physiological situation. WT FLCN was detected by western blot but the mutant protein again showed markedly lower protein levels. This effect was partially reversed by treatment with MG-132, a proteasome inhibitor and chloroquine, suggesting that inhibition of both lysosomal and proteasomal degradation stabilized the mutant protein.

Additionally, fluorescence imaging revealed an altered subcellular localization of mutant FLCN comparing to the WT protein. WT FLCN localized to both cytoplasm and nucleus and the mutant protein was restricted to the cytoplasm. Treatment with MG-132 not only stabilized the mutant protein but also led to a nuclear localization shift making it more similar to the WT. This may be the consequence of accumulation of ubiquitinated mutant FLCN, since ubiquitination has been shown to be a key regulator of subcellular localization of different proteins. It remains to be elucidated whether a ubiquitination or similar approach may have any therapeutic implication in the treatment of BHD – once stabilized and correctly localized – to fix the molecular function required to prevent tumorigenesis.

While this manuscript was under revision a different group characterized the same mutation in two sisters with RCC and found similar results regarding splicing of WT and mutant FLCN (Rossing et al., 2017).

In summary, this study shows that the functional characterization of the pathogenic mutations in BHD syndrome may shed light into further research for the development of novel diagnostic and therapeutic strategies.

  • Bartram MP, Mishra T, Reintjes N, Fabretti F, Gharbi H, Adam AC, Göbel H, Franke M, Schermer B, Haneder S, Benzing T, Beck BB, & Müller RU (2017). Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer. BMC medical genetics, 18 (1) PMID: 28499369

Findacure workshop – “Engaging your community for Fundraising”

Fundraising helps charities to make a difference for rare disease patients by supporting research, community events and awareness campaigns. At the end of April, Findacure hosted a workshop in London with several speakers sharing their experience of fundraising.

Libbie Read and Mary Rose Roberts, from Findacure, introduced the fundraising theme and gave a talk about how to engage your community for fundraising. They mentioned the benefits of community fundraising: the financial side; unrestricted funding; gift aid; raising awareness; educating; engaging new audiences; building support by establishing new relationships and growing existing ones. They gave examples of fundraising events including ones organised by Findacure  – gala dinners, firewalk, London marathon, pub quizzes, etc., and discussed in detail the planning of a fundraising event, dividing it in different stages and suggesting the steps to follow for each stage:

Initial planning:

  • Outline your fundraising idea
  • Identify your stakeholders
  • Define aims and objectives
  • Consider what is achievable
  • Set a budget – prepare to compromise
  • Remember to check the calendar

Implementation:

  • Assign tasks
  • Set a schedule and make note of deadlines
  • Secure funding
  • Promote – marketing and communication is essential
  • Remember to expect the unexpected.

Post-project:

  • Follow up with participants
  • Collect feedback
  • Evaluate
  • Claim Gift Aid

Robin Marshall, from the AKU Society, talked about putting together special fundraising events. He talked about the things to consider when:

 Planning a budget:

  • Equipment hire
  • Prizes
  • Transport
  • Insurance
  • Fees for licenses and permissions
  • Return of investment
  • Hidden costs (have a contingency plan)

Choosing a venue:

  • Meet objectives
  • Fit the audience
  • Travel costs

Organising a team:

  • Staff/volunteers
  • Time commitments
  • Particular expertise needed?
  • Define roles/responsibilities

Callum Appleby from the Bone Cancer Research Trust gave an exciting talk about fundraising with challenge events. He started by highlighting that challenge events make people achieve more than they think it’s possible. They are events that take people out of their comfort zone, it can be anything and it varies from one person to another. Challenge events can be pre invested or own place events. In pre invested events, the charities buy places in these events from either the event organisers or 3rd party’s acting on behalf of the organisers, the costs can vary, buying places means charities have secured places and can ask people to raise sponsorship in return for a place. In own place events there is no investment by charities. People secure their own places in the event then take part for an organisation and the amount a person raises is completely up to them. Callum mentioned the importance of choosing a successful event by asking questions such as:

  • Is the event well established?
  • Does the event sell out?
  • Does it fit a gap in the market?
  • Is the event popular amongst your target demographic?
  • What makes the event special?
  • What is the cost? – Ensure high return
  • Location and support costs

In special eventsrecruitment can be a challenge, so it is important to build a database of contacts, to offer incentives, to have a call to action, to reach new audiences and to explain clearly the need for support.

Sharmila Nikapota from the Sohana Research Fund gave a presentation about special fundraising events for your community such as gala dinners, auctions, etc., that have a high level of entertainment and that make the participants feel special, have a good experience and reward the organisers.

NKTR-214 therapy study in patients with RCC

Early this year at the ASCO Genitourinary Cancers 2017 meeting, Hurwitz et al. (2017) presented clinical data from a Phase I clinical trial of the oncology agent NKTR-214 in patients with renal cell carcinoma (RCC) showing encouraging evidence of anti-tumour activity, and a favourable safety and tolerability profile. NKTR-214 was developed by Nektar Therapeutics to expand specific cancer-fighting immune cells in the tumour environment and increase expression of the cell surface receptor PD-1 on these immune cells.

NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signalling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and natural killer (NK) cells – usually cancer-fighting immune cells – over T regulatory cells (Tregs) – immunosuppressive cells that usually limit anti-tumour response.

A dose escalation trial of NKTR-214 was initiated to assess the safety, tolerability and explore immune changes in the blood and tumour microenvironment in patients with RCC. NKTR-214 was administered IV every 2 or 3 weeks. Pre and post treatment blood and tumour samples were collected and analysed for immune phenotyping, gene expression and changes in the tumour microenvironment by immunohistochemistry.

Among 25 patients dosed, 15 had RCC. Treatment with single-agent NKTR-214 was well tolerated and the maximum tolerated dose (MTD) was not reached. There were no autoimmune-related adverse events or organ related inflammation. 6 out of the 15 RCC patients, with prior tyrosine kinase inhibitor (TKI) treatments, experienced tumour shrinkage. Analysis of the tumour microenvironment revealed several significant immunological changes post treatment, including increase in total and proliferating NK, CD8+, and CD4+ T cells. There was good correlation between increase in activated CD4+ and CD8+ T cells in peripheral blood with an increase in T cell infiltrates within the tumour tissue. There was a greater abundance of CD8+ T cell compared to Treg immune suppressive cells accumulating in the tumour tissue. NKTR-214 also increased cell-surface expression of PD-1 on CD4+ and CD8+ T cells.

NKTR-214 increased immune infiltration in the tumour and anti-tumour activity in patients who previously progressed on TKIs, with a favourable safety profile. The ability to alter the immune environment and increase PD-1 expression on effectors T cells may improve the effectiveness of anti-PD-1 blockade. A trial combination of NKTR-214 and nivolumab is being evaluated. The Phase 1/2 clinical program will enrol up to 260 patients and will evaluate the potential for the combination of Opdivo (nivolumab) and NKTR-214 to show improved and sustained efficacy and tolerability above the current standard of care in melanoma, kidney, triple-negative breast cancer, bladder and non-small cell lung cancer patients.

The NKTR-214 clinical trial is currently recruiting participants, you can find more information here.

Findacure – Medical Research Explained: Clinical research

Following last week’s blog about pre-clinical research, this week we introduce the second part of Findacure’s webinar explaining clinical research.

The subject of clinical research was presented by Sarah Venugopal from Raremark, a company connecting families affected by rare diseases with information on the latest research and treatments in the field.

Clinical research, also known as clinical trials, clinical studies or human trials, are conducted to collect data about the safety and effectiveness of a potential new test, drug or device before it is approved and widely used.

The key players in clinical research are:

  • The sponsor – organization that funds the clinical trial, can be a pharma company, a charity, a hospital or even an individual
  • The ethics review boards – independent groups responsible for the protection of the rights, safety and well-being of people taking part in a trial
  • The principal investigator (PI) – person who leads the trial, usually a specialist doctor or researcher
  • The study coordinator – person who supports the PI and is in charge of the day-to-day running of the trial, there can be more than one
  • The participants – the patients or healthy volunteers taking part in the trial

People take part in clinical trials to help speed up the approval process for new drugs for themselves or others, to access specialist care, to potentially access an experimental treatment and to be monitored closely.

Main types of clinical trial:

  • Diagnostic trials – new tests or procedures for diagnosing diseases
  • Natural history studies – often done for rare diseases, they generate insights into how diseases might progress naturally over time
  • Observational studies – monitor participants without intervening for example whilst they are already on an existing treatment or after surgery
  • Screening trials – evaluate new tests, test the best way to detect certain diseases or medical conditions
  • Treatment trials – the one people are more familiar with, they evaluate the effectiveness and safety of potential new treatments

Four phases of Clinical trials:

Phase I – Typically a small study (5-100) with healthy volunteers assessing safety usually with a very low dose of the drug. This phase usually lasts months to a year. In rare diseases, sometimes this phase is with patients with the condition so that the drug can be approved faster. 70% of the drugs tested on phase I make it to phase II.

Phase II – Assessing safety and efficacy, larger study with people with the condition, can include a placebo. 33% of the drugs tested on phase II make it to phase III.

Phase III – Larger studies, randomized and controlled, people with the condition, lead to a potential approval, looks at dosing. 70-90% of the drugs tested on phase III make it to phase IV.

Phase IV – Drug is approved for use, monitor long-term safety and effectiveness in the real world.

Usually the entire process lasts about 6 to 10 years.

What happens during a trial?

It starts with the sponsor designing the trial with doctors/investigators, then investigators see patients and collected data, the data is entered into a database, the data is analysed and presented in a report and finally the report is used to support approval.

Most large clinical trials are randomized, double-blind placebo-controlled trials. This is a common way to design a trial as it ensures the data is robust, reliable and reduces bias.

Placebo-controlled – a placebo contains no active ingredients and is given to some of the participants in a trial so that the effectiveness of the drug can be accurately seen

Randomized – participants are randomly assigned to either take the active drug or the placebo

Double-blind – to reduce bias, neither the researchers nor the patients know who is taking the experimental drug or the placebo

What happens after a trial?

If the drug is shown to be safe the results are submitted for approval to regulatory bodies (FDA or EMA), this process takes a very long time and the data is reviewed to make sure is reliable, cost-effective and has a positive impact. Additional data might be needed and it important to continue to monitor drug in real world. Usually there is publication of results on PubMed or clinicaltrials.gov but unfortunately, not all sponsors publish their results.

You can watch the entire Findacure webinar here and learn about BHD Syndrome clinical trials here.

Findacure – Medical Research Explained: Pre-clinical research

Last week Findacure hosted a webinar explaining the complex medical research field that introduced the different stages of pre-clinical and clinical research necessary to get a treatment available to patients.

The first talk focused on pre-clinical research and was presented by Oliver Timmis from the AKU Society, a charity that supports patients with the rare condition alkaptonuria.

Oliver explained that assessing a potential treatment of a condition by a drug starts with pre-clinical research. Pre-clinical research in drug development aims to show that a drug is safe and effective in a non-human model before human research is initiated. The drug development timeline is time consuming, taking 16 years on average from the discovery of a drug to having it as an available treatment for patients. The timeline starts with several years of pre-clinical testing and then clinical testing in different phases. Pre-clinical testing is quite variable, compounds are narrowed down at this phase with just a few going to clinical trials – this has very high costs (~$1 billion net cost invested over 15 years).

Pre-clinical development was defined as the testing of promising candidate compounds to ensure that they are:

  • Likely to be effective
  • Safe
  • Sufficiently stable
  • Excreted safely from the body

Around the world there are regulatory guidelines by agencies such as the FDA and the EMA that help in the development of effective pre-clinical and clinical research.

Animal studies are widely used in pre-clinical research, in fact, they are a legal requirement for any human drug developed in Europe.  Animal studies are a standard procedure but controversial, therefore, ethical guidance for appropriate use is in place with systems such as the principles of the 3Rs.

Research can be divided into two categories – Basic and Applied/Clinical. Basic research aims to discover facts about how and why things occur without any relation to clinical outcome; applied research uses information generated by basic research to treat and prevent illness.

You can find BHD-specific basic and clinical research in the BHD Article Library.

Before pre-clinical research there is a period of “pre-pre-clinical research” to establish a target, to choose a molecule that is likely to work, act on symptoms/pathways most important to patients, and work out how to measure if this molecule is having the expected effect.

A classic type of pre-clinical studies is toxicology studies. These studies are usually performed in animals to support human studies. Important considerations are which species and how many animals to use.  It has to be practical and the duration of studies should not be too long since the idea is to get to clinical studies quickly.

Toxicology studies are conducted to assess:

  • Toxic effects following single and multiple dosing
  • Effects on reproduction
  • Potential for teratogenicity
  • Peri/postnatal effects
  • Potential to cause cancer and genetic abnormalities
  • Effects in the immune system
  • Potential to cause skin and eye problems

There are acute/short term toxicity studies which look for effects over short periods using the route of administration intended for humans. Usually, animals are observed for 30 days for eating/drinking habits, weight change, toxic effects and psychomotor changes. Subacute/subchronic studies, uses repeat dosing aligned to intended human usage. Testing over 90-180 days is required to support human administration for 1 week. For a chronic human illness testing over 1 year is required in animals

Definitive animal studies define the No Observable Adverse Effect Level (NOAEL) – the highest amount of drug used with lowest side effects. There is the need to use most sensitive species available and to consider the treatment regime.

An important tool used in medical research is the biomarker. Biomarkers are surrogates for problems in the human body, changes in biological systems that are related to exposure to a toxin. To be useful in pre-clinical studies biomarkers should be chemical specific, quantifiable at low levels, and the ability to be monitored in a non-invasive way is an advantage. Different types of biomarkers include biomarkers of exposure, of response effects and of susceptibility.

In summary, preclinical studies are undertaken to ensure that medicines are safe and effective.  Success in preclinical studies means that a drug has been demonstrated to be probably effective, safe, sufficiently stable, and excreted safely from the body.  Then agreement from the regulators is needed before moving on to clinical research (testing in humans).

Clinical research, the second part of this webinar will be discussed in a future blog.

You can watch the entire webinar here.

DHM attenuates obesity-induced slow-twitch-fiber decrease via FLCN/FNIP1/AMPK pathway

Obesity is often associated with decreases in the proportion of skeletal muscle slow-twitch fibers and insulin sensitivity. Slow-twitch fibers are rich in mitochondria and utilize fatty acid oxidative phosphorylation for energy production. In their new study, Zhou et al. (2017) explore the role of the FLCN/FNIP1/AMPK signalling pathway in obesity-induced reductions in slow-twitch fibers and insulin sensitivity in skeletal muscle using high-fat-diet-induced (HFD) obese mice, ob/ob mutant mice, and palmitate-treated C2C12 myotubes. The authors also assess the effects of dihydromyricetin (DHM) on the obesity-induced decrease in slow-twitch fibers, and the molecular mechanisms responsible for this effect.

AMP-activated protein kinase (AMPK) plays a central role in skeletal muscle oxidative metabolism and fiber-type specification. AMPK activation in skeletal muscle induces expression of its downstream transcriptional regulator PGC-1α. FLCN, responsible for Birt-Hogg Dubé syndrome, interacts with the AMPK signalling pathway by binding to folliculin-interacting protein 1 (FNIP1) (Baba et al., 2006). Folliculin (FLCN) and FNIP1 may regulate skeletal muscle-fiber-type specification through the AMPK/PGC-1α pathway (Hasumi et al., 2012). Although the interaction between FLCN/FNIP1 and AMPK appears to play an important role in skeletal muscle adaptations, its involvement in the obesity-induced decrease in slow-twitch fibers and insulin resistance remains unclear.

Exercise is commonly prescribed for obesity and metabolic diseases, including insulin resistance and diabetes, since it increases AMPK activity, promoting slow-twitch fibers and increasing the use of fatty acids in skeletal muscle (Lantier et al., 2014). The authors have previously reported that the flavonoid DHM enhanced exercise performance (Zou et al., 2014), and improved skeletal muscle insulin resistance by autophagy induction via AMPK (Shi et al., 2015). However, the ability of DHM to increase the proportion of skeletal muscle slow-twitch fibers via the AMPK signalling pathway remains unclear.

In the HFD-fed and ob/ob mice the proportions of slow-twitch fibers, insulin sensitivity (detected by the markers of insulin sensitivity, insulin-stimulated Akt and insulin receptor substrate 1 (IRS-1) phosphorylation) and oxidative metabolism in skeletal muscle were decreased compared with control mice, and this effect was prevented by DHM treatment.

Increased non-esterified fatty acids (NEFA) levels are closely associated with insulin resistance in obesity and type 2 diabetes. In line with these results, the authors found that plasma NEFA levels were significantly increased in HFD-fed and ob/ob mice compared with controls and they negatively correlated with slow-twitch-fiber proportion.

To verify the results obtained in mice, in vitro experiments with C2C12 myotubes were performed. Palmitate, one of the most elevated plasma NEFA in obesity, was used to induce insulin resistance in C2C12 myotubes and shown to decrease expression of slow-fiber specification Myh7 protein, this was inhibited by DHM.

Western blot analysis show decreased phosphorylation of AMPK in both HFD-fed and ob/ob mice, and in palmitate-treated C2C12 myotubes. The similar trends in AMPK activity and changes in slow-twitch fibers and insulin resistance suggest that AMPK might be involved in these obesity-induced changes. FNIP1 and FLCN expression levels were significantly increased in skeletal muscle in HFD-fed and ob/ob mice, and in palmitate-treated C2C12 myotubes and negatively correlated with AMPK activity. These results implicated FNIP1/FLCN in obesity-induced AMPK inactivation, and the subsequent decreases in slow-twitch fibers and insulin sensitivity in skeletal muscle.

The role of the FLCN/FNIP1/AMPK signalling pathway in obesity-induced insulin resistance and the decrease in slow-twitch fibers was further clarified using over-expression and knock-down of FNIP1 and FLCN. Transfection of C2C12 myotubes with FNIP1 resulted in a corresponding increase in FLCN levels. AMPK phosphorylation levels increased following FLCN or FNIP1 knock-down, and decreased following their over-expressions. mRNA levels of the PGC-1α encoding gene were assessed and results showed that its expression was negatively related to FNIP1/FLCN expression, and consistent with AMPK activity.

DHM ameliorated the obesity-induced decrease in slow-twitch-fiber proportion, insulin sensitivity, and AMPK activity. However, it was necessary to verify if these effects of DHM were mediated by FNIP1 and FLCN. FNIP1 and FLCN expression levels were significantly decreased following DHM administration in HFD-fed and ob/ob mice, and in palmitate-induced C2C12 models. Also, the preventive effects of DHM on the palmitate-induced decrease in slow-twitch fibers, AMPK activation, p-Akt and p-IRS-1 expression, were blocked by FLCN over-expression. These results demonstrated that the effects of DHM were mediated by the FNIP1/FLCN/AMPK signalling pathway.

 

Obtained from Zhou et al. (2017)

Yan et al. recently reported FLCN/AMPK as a novel molecular pathway involved in regulating mitochondrial function and browning of white adipocytes, this was discussed on a previous blog. Here, the results of the study demonstrate that the FNIP1/FLCN complex might play an important role in AMPK/PGC-1α signalling in the obesity-induced decreases in slow-twitch-fibers and insulin sensitivity. Furthermore, DHM acts as a potential exercise mimetic by attenuating these obesity-induced effects via the FNIP1/FLCN/AMPK signalling pathway. These results provide new insights into the FNIP1/FLCN/AMPK signalling pathway, key in BHD research, and novel mechanisms and potential targets for treatments of insulin resistance and type 2 diabetes.

  • Zhou Q, Gu Y, Lang H, Wang X, Chen K, Gong X, Zhou M, Ran L, Zhu J, & Mi M (2017). Dihydromyricetin prevents obesity-induced slow-twitch-fiber reduction partially via FLCN/FNIP1/AMPK pathway. Biochimica et biophysica acta PMID: 28363698

Novel FLCN mutations in Chinese patients

The gene FLCN is inactivated in individuals with BHD syndrome. The FLCN gene encodes the protein Folliculin, which is a putative tumour suppressor. Over 150 different FLCN mutations have been identified, most of which are likely to be pathogenic (LOVD-hosted FLCN mutation database). The majority of these mutations are frameshift, nonsense, insertion/deletion, or splice site mutations, resulting in truncation and inactivation of the encoded protein folliculin. FLCN consists of 14 exons spanning approximately 20 kb of genomic DNA (Nickerson et al., 2002).

Novel FLCN mutations are still being identified and here we discuss two case studies with novel mutations found in patients in China:

Li et al. (2017) report two Chinese BHD patients with novel FLCN mutations. The first case was a 54-year-old man with renal cell carcinoma (RCC), spontaneous pneumothorax (and spontaneous pneumothorax in his family history) and no apparent skin lesions. Genetic testing revealed a novel frameshift mutation (c.946-947delAG) of the FLCN gene. The second case was very similar, a 37-year-old man with chromophobe RCC, no cutaneous lesions, history of spontaneous pneumothorax and family history of pneumothorax. Genetic testing revealed the novel mutation c.770-772delCCT.

Hao et al. (2017) report the case of a 56-year-old Chinese woman who presented with multiple skin papules, pneumothorax and multiple bilateral pulmonary cysts. The patient underwent a tube thoracostomy and had a family history of spontaneous pneumothorax, of pulmonary bullae and of renal cell carcinoma (RCC). The patient’s pneumothorax recurred after 12 years and then she developed another pneumothorax at age 50 years. The patient then underwent surgical intervention. A chest computed tomography (CT) scan showed multiple cystic lesions. Ultrasound examination showed that the patient did not develop RCC, but multiple thyroid nodules were spotted (read previous blog about BHD and thyroid conditions). Genetic testing identified four FLCN mutations. An unreported mutation (c.2297 T > C) in exon 14, and three other mutations that were previously reported to have a minimal correlation to the onset of BHD (Cho et al., 2008) – a mutation in exon 1 (c.-299C > T), a mutation in intron 8 (c.871 + 36G > A) and a mutation in intron 9 (c.1062 + 6C > T).

To date, around 90% of the reported BHD patients are from Europe and the United States. The incidence of skin symptoms seems to be lower among Asian BHD patients (~30%) compared with the higher incidence reported among patients from western countries (~90%) (Murakami et al., 2014Toro et al., 2008Furuya et al., 2013). On the other hand, recurrent pneumothorax is observed in approximately 90% of Asian patients compared to the lower percentage among Western countries patients (Kunogi et al., 2010).

In patients with RCC and pulmonary cysts but without cutaneous lesions, screening for mutations in the FLCN gene should be performed, especially for those with a family history of RCC or pneumothorax. Novel FLCN mutations are still being identified all over the world, to add to those already listed in LOVD-hosted FLCN mutation database. Further studies using large cohorts are still needed to clarify possible genotype–phenotype correlations in BHD syndrome.

  • Li T, Ning X, He Q, & Gong K (2017). Novel germline mutations in FLCN gene identified in two Chinese patients with Birt-Hogg-Dubé syndrome. Chinese journal of cancer, 36 (1) PMID: 28069055
  • Hao S, Long F, Sun F, Liu T, Li D, & Jiang S (2017). Birt-Hogg-Dubé syndrome: a literature review and case study of a Chinese woman presenting a novel FLCN mutation. BMC pulmonary medicine, 17 (1) PMID: 28222720

BHD syndrome: a new case report and a review

Birt-Hogg-Dubé syndrome (BHD), also known as Hornstein–Knickenberg syndrome is an inherited disease associated with skin lesions, lung cysts, pneumothorax and kidney cancer.

Jensen et al. (2017) present a new case report of BHD and a review of the literature. This is a great opportunity to review the genetics, clinical manifestations, diagnosis, treatment, prognosis and follow-up strategies and to draw attention to BHD, unknown to many physicians. Early diagnosis is crucial so that patients can have access to systematic screening for kidney cancer.

The new case report is of a 29-year old female presenting with a spontaneous pneumothorax (SP) two days after running a marathon. The patient also had 11 relatives with cases of SP, therefore she was referred for follow-up. A high-resolution computed tomography (HRCT) scan showed multiple cysts in the lungs, which led to the suspicion of BHD. Genetic testing revealed a mutation (c.1285delC) in the FLCN gene which confirmed the diagnosis of BHD. Family members were offered genetic counselling and 11 family members were diagnosed. The patient and her affected family members were offered a follow-up program with MRI of the kidneys and pulmonary function tests.

Obtained from Jensen et al. (2017)

A search on PubMed and Embase with the terms ‘Birt-Hogg-Dubé syndrome’ and ‘Hornstein-Knickenberg syndrome’ identified 330 papers. Additional articles were identified from reference lists of the already identified papers (snow ball search).

The authors then reviewed what is known about BHD based on these publications, some of which we highlight here:

  • BHD is named after 3 Canadian doctors, who described the syndrome in 1977. They reported a family of 70 members with 15 family members who developed skin lesions on the face, neck, and torso.
  • The autosomal dominant inheritance of the combination of skin manifestations and renal cell carcinoma (RCC) in BHD was first described by Toro et al. in 1999. In this study, lung manifestations were also noticed.
  • In 2001, Schmidt et al. and Khoo et al. located the gene locus of BHD to be on the short arm of chromosome 17 and in 2002 Nickerson et al. linked BHD to the FLCN gene on chromosome 17, which encodes the protein folliculin.
  • Zbar et al. (2002) reported that patients with BHD had a 50-fold risk of SP and a 7-fold increased risk of developing RCC. The histology of renal tumours in BHD is diverse.
  • Several FLCN interacting proteins including FNIP1 and FNIP2 have been identified (Baba et al., 2005; Hasumi et al., 2008).
  • FLCN regulates numerous signalling pathways, including the mammalian target of rapamycin (mTOR) pathway.
  • Neither gender, nor smoking, nor other risk factors have been reported as predictors of the development of cysts or SP. Lung function is rarely affected.
  • Almoosa et al. (2006) found that pleurodesis decreases the pneumothorax recurrence rate, and pleurodesis after the first SP in BHD has been recommended.
  • Skin manifestations are common in BHD and are seen in approximately 58–90% of patients. Fibrofolliculomas are the most frequent, but also trichodiscomas and acrochordons have been described. Fibrofolliculomas present as multiple, pale yellow or white, slightly elevated, dome-shaped, and smooth tumours with a diameter of 2–4 mm. Fibrofolliculomas are predominantly located in the face, neck, and upper torso.
  • A recent double-blind placebo-controlled randomized clinical trial showed no effect of the topical mTOR inhibitor rapamycin on fibrofolliculomas in BHD patients.
  • Since 2008, six BHD symposia have been held where researchers, clinicians, and patients meet and discuss the progress in the BHD field.
  • Currently, the BHD foundation is aware of over 600 BHD families worldwide.
  • Due to its rarity and to the variable presentation of the symptoms, the diagnosis of BHD is often delayed for years. The European BHD consortium has proposed a set of criteria for the diagnosis of BHD. Upon diagnosis of BHD, the patients should undergo examination of the skin, CT imaging of the chest, abdominal MR or CT imaging for renal tumours as well as genetic screening for pathogenic FLCN mutations.

In summary, knowledge in the BHD field is still limited. The variable clinical manifestations of BHD and the fact that no genotype-phenotype correlations have been found, makes early diagnosis and management of BHD complex. Further research is needed to investigate the exact mechanisms of pathogenesis and to optimize the management of BHD patients.

  • Jensen, D., Villumsen, A., Skytte, A., Madsen, M., Sommerlund, M., & Bendstrup, E. (2017). Birt–Hogg–Dubé syndrome: a case report and a review of the literature European Clinical Respiratory Journal, 4 (1) DOI: 10.1080/20018525.2017.1292378

Ammonium regulates mTOR signalling

mTORC1 and mTORC2 are two distinct mammalian TOR (target of rapamycin) complexes that regulate cell growth and metabolism. In cancer, genetic alterations lead to activation of mTOR signalling impacting tumour metabolism. Upregulated glutaminolysis is part of the metabolic reaction occurring in cancer that liberates high levels of ammonium, a toxic waste product. Although the importance of glutamine as a tumour nutrient is recognized, little is known about the potential effects of ammonium produced by glutaminolysis in tumours. In their new study,  Merhi et al., 2017 identify ammonium as a dose-dependent regulator of mTORC2, mTORC1 and of proliferation in cancer cells.

To study potential signalling pathways responding to ammonium, the authors performed a kinase array with lysates of MCF-7 breast cancer cells and detected an increase in the phosphorylation of the kinases AKT-S473 and ERK1/2 upon ammonium addition in a time and dose dependent manner. This was confirmed by western blot. Similarly, the authors observed AKT phosphorylation in other cancer and fibroblast cell lines. The upstream pathway leading to the induction of AKT phosphorylation was investigated. Pre-treatment of MCF-7 cells with a PI3K inhibitor impaired AKT phosphorylation and prevented its induction by ammonium supplementation. mTORC2 has been shown to promote cell proliferation and survival by phosphorylation and activation of the AKT and SGK and the phosphorylation of AKT-S473 is a good readout for mTORC2 activity (Oh et al., 2011). Merhi et al. show that siRNA-mediated knockdown of RICTOR (a subunit of mTORC2), leads to reduction of basal and ammonium-induced AKT-S473 phosphorylation. In addition, ammonium also induced NDRG1-T346 phosphorylation, another readout of mTORC2 activity. Knockdown of YES1 kinase and pharmacological inhibition of the focal adhesion kinase (FAK) decreased ammonium-induced AKT-S473 and NDRG1-T346 phosphorylation. The authors also addressed if integrins, known regulators of FAK signalling, was involved in the ammonium-induced AKT-S473 phosphorylation. Results showed that knockdown of ITGβ1 decreased the basal and the ammonium-induced AKT-S473 phosphorylation. Collectively the data indicates that ammonium-induced activation of mTORC2 involves ITGβ1, FAK, YES1, and PI3K signalling.

Ammonium treatment has been shown to induce a transient increase in calcium in cultured astrocytes (Rose et al., 2005). To explore this, the authors assessed the role of calcium in ammonium-induced mTORC2 activation. Pre-treatment of cells with a calcium chelator decreased basal and ammonium-induced AKT-S473 and NDRG1-T346 phosphorylation. In addition, an increase in cytoplasmic calcium concentration induced a rapid increase in AKT-S473 phosphorylation in a mTORC2- dependent way, suggesting that ammonium-induced mTORC2-dependent AKT phosphorylation is modulated by intracellular calcium mobilization.

The impact of ammonium on the activity of mTORC1 remains unclear. The kinase array revealed a decreased phosphorylation of p70S6K-T389, an mTORC1 readout, after treatment with ammonium, suggesting ammonium potentially inhibits mTORC1. Western blot analysis confirmed this mild decrease in phosphorylation. However, this weak dephosphorylation appeared transient, suggesting that the inhibition of mTORC1 was quickly compensated by a reactivation. AKT activation has been shown to promote mTORC1 by inhibiting the TSC complex. In addition, AKT-mediated phosphorylation of another negative regulator of mTORC1, PRAS40, prevents its inhibitory role (Dibble et al., 2015). Merhi et al. found that ammonium induced the rapid phosphorylation of TSC2-T1462, PRAS40-T246 and of 4EBP1 – another readout of mTORC1 activity – suggesting consequent rapid mTORC1 activation. This shows an additional regulatory process upon ammonium addition that transiently counteracts the mTORC1-mediated stimulation of p70S6K-T389 phosphorylation. Treatment with an inhibitor of AKT, not only prevented the ammonium-induced phosphorylation of AKT, but also reduced the phosphorylation of TSC2, PRAS40 and 4EBP1, consistent with ammonium-induced activation of mTORC1 being AKT-dependent.

The impact of ammonium on the proliferation of MCF-7 cells was also assessed. Adding high concentrations of ammonium (~15 mM) resulted in significant cell growth inhibition while low concentrations stimulated growth both in the presence or absence of glutamine supplementation.

In summary, the authors show that ammonium triggers mTORC2-dependent phosphorylation of AKT-S473 in cancer cells. The mTORC2 activation occurs via the PI3K pathway and relies on YES1 and FAK kinases, on integrin ITGβ1 and on intracellular calcium stores mobilization. In addition, ammonium also leads to an AKT-dependent stimulation of mTORC1 signalling and to a dose-dependent stimulation of proliferation. This study identifies that ammonium, a waste product of cancer cells, impacts both mTORC2 and mTORC1 signalling and brings insights into the molecular mechanism of the ammonium-mediated regulation and tumour growth.

FLCN, the gene responsible for BHD Syndrome, has been shown to regulate mTOR signalling. Our interactive folliculin signalling diagram illustrates the relationship between Folliculin (FLCN) and several proteins and signalling pathways including mTORC1 and mTORC2.

  • Merhi A, Delrée P, & Marini AM (2017). The metabolic waste ammonium regulates mTORC2 and mTORC1 signaling. Scientific reports, 7 PMID: 28303961