RANK and RANKL link female sex hormones to BRCA1 mutation-induced breast cancer.
Although most breast cancers arise sporadically, approximately 5-10% of breast cancers are inherited through germline mutations. Among the inherited breast cancer cases, the overwhelming majority are caused by mutations in the tumor suppressor genes BRCA1 or BRCA2. The lifetime risk of developing breast cancer for BRCA1-mutation carriers is up to 85%, and up to 66% for BRCA2-mutation carriers. BRCA1 mutation carriers often opt for bilateral mastectomy surgeries and prophylactic salpingo-oophorectomies, though these actions are often associated with wide-ranging risks and psychological as well as psychosocial effects. Therefore, the search for an alternative, non-invasive prevention strategy is of paramount importance for many women carrying BRCA mutations, as well as for other women with increased risk of breast cancer. Exposure to endogenous or exogenous hormones is a significant risk factor for breast cancer development. Several studies have clearly demonstrated a strong relationship between sex hormones and breast cancer.
RANK and RANKL are key molecules downstream of steroid hormones in the mammary gland, and we have discussed previously that breast cancer development in BRCA1 mutation carriers is crucially regulated by female sex hormones (Schramek et al. Nature 2006). It is intriguing to question whether the RANKL/RANK pathway has a role in the etiology of BRCA1 mutation-driven breast cancer. Indeed, e recently we provided direct genetic evidence for the function of RANK/RANKL in driving Brca1 mutation-driven breast cancer (Sigl et al. Cell Res. 2016). Mice carrying Brca1;p53 mutations exhibited excessive proliferation and malignancy in their mammary glands at the age of 4 months. Intriguingly, genetic concurrent deletion of Rank in these mice significantly decreased this excessive proliferation and almost completely abrogated the occurrence of malignancy. Moreover, the colony forming capacity of human mammary epithelial progenitor cells from heterozygous BRCA1-mutation carriers can be significantly inhibited by treatment with Denosumab, the monoclonal antibody of RANKL. In addition, we detected high RANK expression in pre-malignant lesions and breast cancer samples of patients carrying a BRCA1-mutation. Finally, with access to data from the Collaborative Oncological Gene-environment Study (iCOGS), which includes approximately 15,200 BRCA1 and 8,200 BRCA2 mutation carriers, we found six SNPs in the TNFRSF11A locus (TNFRSF11A encodes RANK) that were significantly associated with breast cancer risk in the overall series of BRCA1 mutation carriers. Taken together we have shown that RANKL/RANK act on both murine and human mammary progenitor cells, that RANKL/RANK are indeed critical regulators for the initiation, as well as progression, of BRCA1 mutation-driven mammary cancer, and, most importantly, the pathway offers a new, less-invasive therapeutic target for women genetically predisposed to BRCA1-driven breast cancers.
Licensing Natural Killer cells to kill cancer metastases.
Tumor metastasis is the primary cause of mortality in cancer patients, and constitutes a major challenge in cancer therapy. The immune system is not only responsible for controlling infection, but also for recognizing and destroying cancer cells. We showed that deletion or targeted inactivation of the E3 ligase Cbl-b efficiently enhances the anti-tumor function of NK (Natural Killer) cells. As a result, the progression of metastases in melanoma and breast cancer was significantly inhibited. We then identified a molecular pathway by which Cbl-b blocks NK cell activity towards metastatic tumors, namely via a family of molecules known as TAM receptors. We developed an inhibitor directed against TAM receptors, and found that blocking these receptors through different routes of application, including via an oral ‘pill’, markedly reduced metastatic spread in two model systems. We further showed that the widely used anticoagulant warfarin exerts anti-metastatic cancer activity in mice by blocking Cbl-b/TAM receptors in NK cells, thus providing a molecular explanation for the enigmatic role of warfarin in reducing cancer metastases reported more than 50 years ago. This novel TAM/Cbl-b inhibitory pathway holds promise for targeted immunotherapy of cancer metastases. Paolino et al. Nature 2014.
Blocking "self-protection" of tumor cells: a novel anti-cancer therapy.
Autophagy is a “recycling programme” of the cells. During this process, endogenous proteins are digested and reused, among other things. This takes place particularly under stressful conditions, and if the supply of nutrients is insufficient - such as in various cancers. This mechanism helps cancer cells survive in rapidly growing tumours, when the energy supply is low. We have shown recently that tissue-specific inactivation of the autophagy gene Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancers, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response (Rao et al. Nat. Commun. 2014). Our results indicate that Atg5-dependent autophagy plays a dual role in lung cancer. Disabled autophagy initially increases the number of tumour foci and accelerates the transition from hyperplasia to adenomas. However, autophagy is then required for the efficient progression from adenoma to adenocarcinoma, indicating that disabled autophagy ultimately reduces tumour mass and improves survival of tumour-bearing mice. We found that disabled autophagy favours adenosinergic signaling via a Hif1α pathway, and the infiltration of tumours by Tregs, thus influencing inflammatory and immunosurveillance mechanisms that can stimulate and control carcinogenesis, respectively. This complex ‘autophagic’ dichotomy may have far-reaching implications for the avoidance and treatment of cancers that would, respectively, benefit from the induction and inhibition of autophagy.
Complete cardiac regeneration in a mouse model of myocardial infarction.
Each year, 17 million people around the world die of cardiovascular diseases, 2 million of them in the EU alone (WHO). Even though medical care for cardiac patients has improved tremendously and the immediate fatality rate has dropped, most patients still face permanent damage leading to chronic heart failure. During a heart attack, cardiac muscle cells die and are replaced by scar tissue. But scar tissue cannot pump, which leads to limitations in cardiac function and a weakening of the heart muscle. So far, heart muscle cells lost in adults cannot be efficiently regenerated, despite innovative approaches such as stem cell therapy. Pioneering experiments demonstrated that fish can completely regenerate the heart following resection of the heart apex, spurning a plethora of studies using fish as a model organism. However, it remained unclear whether the mammalian heart is also able to regenerate after a complex cardiac ischemic injury. We therefore established a protocol to induce a severe heart attack in 1-day-old mice using ligation of the left anterior descending artery (LAD) (Haubner et al. 2012). LAD ligation caused substantial cardiac injury in the left ventricle, as manifested by caspase 3 activation and massive cell death. Ischemia-induced cardiomyocyte death was also seen on day 4 after LAD ligation. Remarkably, within 7 days after the initial ischemic stroke, we observed complete cardiac regeneration with no signs of tissue damage or scarring. This tissue regeneration translated into long-term normal cardiac function, as evidenced by echocardiography. In contrast, LAD ligations in older, 7-day-old mice resulted in extensive scarring, similar to that in adult mice, indicating that the regenerative capacity for complete cardiac healing after a heart attack can be traced to the first week after birth.
First evidence of complete cardiac regeneration in humans.
Our group has recently reported complete morphologic and functional cardiac repair in newborn mice following severe myocardial infarction. Two key issues remain to translate findings in model organisms to future therapies in humans: what is the mechanism, and can cardiac regeneration indeed occur in newborn humans? We now report the case of a newborn child suffering from a severe myocardial infarction due to coronary artery occlusion. The child developed massive cardiac damage as defined by serum markers for cardiomyocyte cell death, electrocardiograms, echocardiography, and cardiac angiography. Remarkably, within weeks after the initial ischemic insult, we observed functional cardiac recovery, which translated into long-term normal heart function. These data indicate that, similar to neonatal rodents, newborn humans have the intrinsic capacity to repair myocardial damage and completely recover cardiac function (Haubner et al. Circ. Research 2015).
Molecular regulation of blood vessel morphogenesis.
The growth of blood vessels is critical for development, tissue regeneration and many pathologies such as cancer and cardiovascular diseases. To understand the molecular networks that control the expansion of blood vessel networks, and to reveal novel targets to either enhance or inhibit angiogenesis, we take advantage of our in-house HAPLOBANK, a biobank of 100,000 individual haploid murine embryonic stem cell lines targeting 16,950 genes. We have developed an assay in which these haploid stem cells can be efficiently differentiated into endothelial networks in culture as well as in teratomas, and can therefore be used to functionally uncover the contribution of genes to phenotypes such as diminished or excessive blood vessel growth. Our long-term goal is to identify molecular switches that allow stimulation of blood vessel growth in ischemic diseases such as heart attack or stroke, as well as to normalize the excessive vessel networks in cancer or diabetic retinopathy.
The RNA kinase CLP1 links tRNA metabolism to progressive motor neuron loss.
CLP1 was the first mammalian RNA kinase discovered, by Javier Martinez at IMBA (Weitzer & Martinez, Nature 2007). However, its in vivo function was elusive. We joined forces with Javier’s lab to generate kinase-dead Clp1 (Clp1K/K) mice. Amazingly, these mice exhibit a progressive loss of spinal motor neurons, along with axonal degeneration in peripheral nerves and denervation of neuromuscular junctions, ultimately resulting in impaired motor function, muscle weakness, paralysis, and fatal respiratory failure. Mechanistically, the loss of CLP1activity leads to the accumulation of an entirely novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-tRNA. These tRNA fragments sensitize cells to oxidative stress-induced p53 activation and p53-dependent cell death. Genetic inactivation of p53 rescued Clp1K/K mice from motor neuron loss, muscle denervation, and respiratory failure. Finally, transgenic rescue experiments re-expressing wild type CLP1 using the Hb9 promoter confirmed that CLP1 must function in motor neurons. Thus, our experiments have uncovered a mechanistic link between tRNA processing, the formation of a new RNA species, and progressive loss of lower motor neurons regulated by p53. (Hanada, et al. Full Article in Nature 2013).
Identification of a new neurological syndrome in children.
In collaboration with Jim Lupski’s lab (Texas), genome analyses identified a CLP1 homozygous missense mutation (R140H) in five unrelated families. The affected individuals develop severe motor and sensory defects as well as cerebral dysgenesis, and exhibit microcephaly. Importantly, using 15.2 Tesla MRI imaging, microcephaly and reduced cortical brain volumes were confirmed in mice that carry a kinase-dead CLP1. The CLP1 mutation in patients causes disrupts the CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex and reduces pre-tRNA cleavage activity, resulting in the accumulation of linear tRNA introns. These data elucidated a novel neurological syndrome affecting both, the PNS and CNS, defined by CLP1mutations that impair tRNA splicing. Kacara et al. Cell 2014.
A novel and critical transcriptional regulator of pain perception.
Chronic and acute pain affects millions of people worldwide, leading to enormous burdens on finances and quality-of-life. The detection of noxious or damaging stimuli (nociception) is an ancient process that alerts living organisms to environmental dangers. Pain perception is essential for an animal to thrive, and human patients that cannot sense pain, such as patients with hereditary sensory and autonomic neuropathy (HSAN), die prematurely due to multiple injuries. PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. We showed that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed that they altered evolutionarily conserved residues. In Drosophila, we identified Hamlet as the functional PRDM12 homologue that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. These data show that PRDM12 and its functional fly homologue Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception (Nagy et al. Cell Cycle 2015).
Genetics of anti-fungal immune responses and novel therapeutic interventions.
Systemic fungal infections claim an estimated 1.5 million lives every year. Immune responses to fungal infections are characterized by an intricate interplay between a multitude of immune cells that aim to eradicate the pathogen, and also to limit pathogen or immune reaction-induced damage to the host. The aim of this project is to both increase our understanding of the genetic basis of anti-fungal immune responses and to define regulatory pathways that suppress the development of protective immunity.
To study the genetic basis of anti-fungal immune reactions we collaborate with human geneticists and develop and establish mouse models for rare human immune disorders. Amongst others, we developed a mouse model for a form of severe congenital neutropenia, a disorder characterized by a severe reduction in the number of neutrophil granulocytes that causes recurrent and often life-threatening infections. In these patients, mutations of JAGN1 cause an absence or dysfunction of neutrophil granulocytes, rendering patients susceptible to pathogens. Using mouse modeling, we determined how loss of this gene affects an immune response on a systemic, cellular and molecular level. Notably, in both human JAGN1-mutant and mouse Jagn1-null neutrophils, we found defective signaling via the G-CSF receptor, whereas signaling via the GM-CSF receptor was apparently normal. In vivo treatment with GM-CSF protected Jagn1-mutant mice from lethality after septic Candida albicans infections. Importantly, our findings in mice translated to humans: in vitro GM-CSF treatment completely restored the defective fungicidal function of bone marrow cells from JAGN1-mutant individuals toward Candida albicans. Thus, our mouse studies have allowed us to uncover a viable treatment option for JAGN1-mutant humans that now needs to be tested in clinical trials.(Wirnsberger et al. Nature Genetics 2014).
Immune responses to fungal pathogens are tightly regulated to prevent immune pathology and tissue damage caused by excessive inflammation. While such regulatory feedback loops are necessary to keep immune responses under control, a carefully tailored enhancement of immune responses holds great promise for treating both infectious and malignant diseases. We have identified the immune regulatory gene Cblb as the first negative regulator of anti-fungal immune responses. Mice lacking this gene were protected from morbidity and mortality caused by systemic and cutaneous fungal infections, due to their ability to mount an enhanced anti-fungal immune response. Mechanistically, we showed that Cblb regulates the recognition of fungal pathogens via tuning the availability and signaling strength of a class of C-type lectin receptors essential for anti-fungal immunity. To exploit this finding, we designed and engineered a peptide-based inhibitor for Cblb to treat systemic fungal infections. Treatment with this novel Cblb inhibitor enhanced the anti-fungal immune response and protected mice from an otherwise lethal fungal sepsis (Wirnsberger et al. Nature Genetics 2016).
RANKL – RANK signaling in osteoclastogenesis and bone disease.
Bone is a tissue which is continuously being rebuilt and remodeled. Osteoblasts are the bone-building cells that deposit new bone tissue, whereas osteoclasts are the bone-resorbing cells that are responsible for breaking down bone. Through a delicate balance between these two cell types, the skeleton is subjected to continuous change. We have shown in the lab through genetic manipulation that RANK (TNFRSF11A) and RANKL (TNFSF11), a receptor-ligand pair of the tumor necrosis factor (TNF) receptor superfamily, are key regulators in bone physiology controlling osteoclast development (Kong et al. Nature 1999, Wada et al. 2006). Osteoprotegerin (OPG) serves as a decoy receptor for RANKL and therefore can inhibit osteoclast development. We have shown that OPG expression is strongly affected by estrogen levels, thereby linking sex hormones to bone turnover. This is of clinical relevance for postmenopausal women whom frequently suffer from bone loss, which is referred to as postmenopausal osteoporosis. Mechanistically we have demonstrated that the loss of estrogen leads to decreased OPG levels and thus a relative rise of RANKL activity, resulting in increased osteoclast numbers and ultimately to enhanced bone turnover and osteoporosis, which is a significant health problem especially in elderly populations. Importantly, Denosumab, a human monoclonal RANKL-blocking antibody, has been developed and approved for the treatment of osteoporosis and skeletal-related events in cancer and has already been benefiting thousands of patients.