Rachel Morissette

sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

A mutation in TGFB3 associated with a syndrome of low muscle mass, growth retardation, distal arthrogryposis and clinical features overlapping with marfan and loeys–dietz syndrome

The transforming growth factor β (TGF‐β) family of growth factors are key regulators of mammalian development and their dysregulation is implicated in human disease, notably, heritable vasculopathies including Marfan (MFS, OMIM #154700) and Loeys–Dietz syndromes (LDS, OMIM #609192). We described a syndrome presenting at birth with distal arthrogryposis, hypotonia, bifid uvula, a failure of normal post‐natal muscle development but no evidence of vascular disease; some of these features overlap with MFS and LDS. A de novo mutation in TGFB3 was identified by exome sequencing. Several lines of evidence indicate the mutation is hypomorphic suggesting that decreased TGF‐β signaling from a loss of TGFB3 activity is likely responsible for the clinical phenotype. This is the first example of a mutation in the coding portion of TGFB3 implicated in a clinical syndrome suggesting TGFB3 is essential for both human palatogenesis and normal muscle growth.

Clinical and biochemical profiles suggest fibromuscular dysplasia is a systemic disease with altered TGF-β expression and connective tissue features

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor β (TGF‐β) cytokines in patient‐derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra‐arterial pathology included low bone density (P<0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF‐β1 (P=0.009), TGF‐β2 (P= 0.004) and additional inflammatory markers, and increased TGF‐β1 (P=0.0009) and TGF‐β2 (P=0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age‐ and gender‐matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF‐β signaling and offers TGF‐β as a marker of FMD.—Ganesh, S. K., Morissette, R., Xu, Z., Schoenhoff, F., Griswold, B. F., Yang, J., Tong, L., Yang, M.‐L., Hunker, K., Sloper, L., Kuo, S., Raza, R., Milewicz, D. M., Francomano, C. A., Dietz, H. C., Van Eyk, J., McDonnell, N. B. Clinical and biochemical profiles suggest fibromuscular dysplasia is a systemic disease with altered TGF‐β expression and connective tissue features. FASEB J. 28, 3313–3324 (2014). www.fasebj.org

Tenascin-X Haploinsufficiency Associated with Ehlers-Danlos Syndrome in Patients with Congenital Adrenal Hyperplasia

CONTEXT The gene for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, CYP21A2, is flanked by the gene encoding tenascin-X (TNXB), a connective tissue extracellular matrix protein that has been linked to both autosomal dominant and autosomal recessive Ehlers-Danlos syndrome (EDS). A contiguous deletion of CYP21A2 and TNXB has been described. OBJECTIVE The objective of the study was to determine the frequency and clinical significance of TNXB haploinsufficiency in CAH patients. DESIGN, SETTING, AND PARTICIPANTS A total of 192 consecutive unrelated CAH patients being seen as part of an observational study at the National Institutes of Health Clinical Center (Bethesda, MD) were prospectively studied during 2006-2010. Patients were evaluated for clinical evidence of EDS, including cardiac evaluation. DNA was analyzed by PCR, multiplex ligation-dependent probe amplification, Southern blot, and TNXB sequencing. Tenascin-X expression was evaluated by Western blot analysis of fibroblasts and immunostaining of the skin. CAH patients with TNXB haploinsufficiency were compared with age-matched CAH patients with normal TNXB (controls). Phenotyping of 7 parents with TNXB haploinsufficiency was performed. MAIN OUTCOME MEASURES The frequency of TNXB haploinsufficiency among CAH patients and the frequency of EDS symptomatology among CAH patients with TNXB haploinsufficiency and controls. RESULTS TNXB haploinsufficiency, here termed CAH-X syndrome, was present in 7% of CAH patients. Twelve of 91 patients carrying a CYP21A2 deletion (13%) carried a contiguous deletion that extended into TNXB. One patient carried a TNXB premature stop codon. Twelve of 13 patients with CAH-X had EDS clinical features. Patients with CAH-X were more likely than age-matched controls to have joint hypermobility (P < .001), chronic joint pain (P = .003), multiple joint dislocations (P = .004), a structural cardiac valve abnormality by echocardiography (P = .02), and reduced tenascin-X expression by Western blot and immunostaining. A subset of parents had clinical findings. CONCLUSIONS Clinical evaluation for connective tissue dysplasia should be routinely performed in CAH patients, especially those harboring a CYP21A2 deletion.

Broadening the Spectrum of Ehlers Danlos Syndrome in Patients With Congenital Adrenal Hyperplasia

CONTEXT The contiguous gene deletion syndrome (CAH-X) was described in a subset (7%) of congenital adrenal hyperplasia (CAH) patients with a TNXA/TNXB chimera, resulting in deletions of CYP21A2, encoding 21-hydroxylase necessary for cortisol biosynthesis, and TNXB, encoding the extracellular matrix glycoprotein tenascin-X (TNX). This TNXA/TNXB chimera is characterized by a 120-bp deletion in exon 35 and results in TNXB haploinsufficiency, disrupted TGF-β signaling, and an Ehlers Danlos syndrome phenotype. OBJECTIVE The objective of the study was to determine the genetic status of TNXB and resulting protein defects in CAH patients with a CAH-X phenotype but not the previously described TNXA/TNXB chimera. Design, Settings, Participants, and Intervention: A total of 246 unrelated CAH patients were screened for TNXB defects. Genetic defects were investigated by Southern blotting, multiplex ligation-dependent probe amplification, Sanger, and next-generation sequencing. Dermal fibroblasts and tissue were used for immunoblotting, immunohistochemical, and coimmunoprecipitation experiments. MAIN OUTCOME MEASURES The genetic and protein status of tenascin-X in phenotypic CAH-X patients was measured. RESULTS Seven families harbor a novel TNXB missense variant c.12174C>G (p.C4058W) and a clinical phenotype consistent with hypermobility-type Ehlers Danlos syndrome. Fourteen CAH probands carry previously described TNXA/TNXB chimeras, and seven unrelated patients carry the novel TNXB variant, resulting in a CAH-X prevalence of 8.5%. This highly conserved pseudogene-derived variant in the TNX fibrinogen-like domain is predicted to be deleterious and disulfide bonded, results in reduced dermal elastin and fibrillin-1 staining and altered TGF-β1 binding, and represents a novel TNXA/TNXB chimera. Tenascin-X protein expression was normal in dermal fibroblasts, suggesting a dominant-negative effect. CONCLUSIONS CAH-X syndrome is commonly found in CAH due to 21-hydroxylase deficiency and may result from various etiological mechanisms.

Transforming growth factor-β (TGF-β) pathway abnormalities in tenascin-X deficiency associated with CAH-X syndrome

Patients with congenital adrenal hyperplasia (CAH) with tenascin-X deficiency (CAH-X syndrome) have both endocrine imbalances and characteristic Ehlers Danlos syndrome phenotypes. Unlike other subtypes, tenascin-X-related Ehlers Danlos syndrome is caused by an extracellular matrix protein deficiency rather than a defect in fibrillar collagen or a collagen-modifying enzyme, and the understanding of the disease mechanisms is limited. We hypothesized that transforming growth factor-β pathway dysregulation may, in part, be responsible for connective tissue phenotypes observed in CAH-X, due to this pathways known role in connective tissue disorders. Fibroblasts and direct tissue from human skin biopsies from CAH-X probands and age- and sex-matched controls were screened for transforming growth factor-β biomarkers known to be dysregulated in other hereditary disorders of connective tissue. In CAH-X fibroblast lines and dermal tissue, pSmad1/5/8 was significantly upregulated compared to controls, suggesting involvement of the bone morphogenetic protein pathway. Additionally, CAH-X samples compared to controls exhibited significant increases in fibroblast-secreted TGF-β3, a cytokine important in secondary palatal development, and in plasma TGF-β2, a cytokine involved in cardiac function and development, as well as palatogenesis. Finally, MMP-13, a matrix metalloproteinase important in secondary palate formation and tissue remodeling, had significantly increased mRNA and protein expression in CAH-X fibroblasts and direct tissue. Collectively, these results demonstrate that patients with CAH-X syndrome exhibit increased expression of several transforming growth factor-β biomarkers and provide a novel link between this signaling pathway and the connective tissue dysplasia phenotypes associated with tenascin-X deficiency.

Ehlers–Danlos Syndrome Caused by Biallelic TNXB Variants in Patients with Congenital Adrenal Hyperplasia

Some variants that cause autosomal‐recessive congenital adrenal hyperplasia (CAH) also cause hypermobility type Ehlers–Danlos syndrome (EDS) due to the monoallelic presence of a chimera disrupting two flanking genes: CYP21A2, encoding 21‐hydroxylase, necessary for cortisol and aldosterone biosynthesis, and TNXB, encoding tenascin‐X, an extracellular matrix protein. Two types of CAH tenascin‐X (CAH‐X) chimeras have been described with a total deletion of CYP21A2 and characteristic TNXB variants. CAH‐X CH‐1 has a TNXB exon 35 120‐bp deletion resulting in haploinsufficiency, and CAH‐X CH‐2 has a TNXB exon 40 c.12174C>G (p.Cys4058Trp) variant resulting in a dominant‐negative effect. We present here three patients with biallelic CAH‐X and identify a novel dominant‐negative chimera termed CAH‐X CH‐3. Compared with monoallelic CAH‐X, biallelic CAH‐X results in a more severe phenotype with skin features characteristic of classical EDS. We present evidence for disrupted tenascin‐X function and computational data linking the type of TNXB variant to disease severity.

Revisiting the prevalence of nonclassic congenital adrenal hyperplasia in US Ashkenazi Jews and Caucasians

PurposeNonclassic 21-hydroxylase deficiency, a mild form of congenital adrenal hyperplasia (CAH), is estimated to be the most common autosomal recessive condition, with an especially high prevalence in Ashkenazi Jews (3.7% affected, 30.9% carriers), based on a 1985 HLA-B linkage study of affected families. Affected individuals, especially women, may suffer from hyperandrogenism and infertility. State-of-the-art genetic studies have not been done to confirm these remarkable rates.MethodsCYP21A2 genotyping was performed in 200 unrelated healthy Ashkenazi Jewish subjects and 200 random US Caucasians who did not self-identify as a specific ethnicity using multiplex minisequencing, real-time polymerase chain reaction and junction site analysis.ResultsNonclassic CAH carriership was found similarly in 15% (95% confidence interval (CI): 10.4–20.7) of Ashkenazi Jews and 9.5% (95% CI: 5.8–14.4) of Caucasians (P=0.13). The proportion of Ashkenazi Jewish nonclassic CAH carriers (0.15 versus 0.309, P<0.0001) and disease affected (0.005 versus 0.037, P=0.009) was not as high as previously reported. The estimated prevalence of nonclassic CAH in Caucasians was 1 in 200 (0.5%, 95% CI: 0.01–2.8).ConclusionNonclassic CAH is a common condition, regardless of ethnicity, and should be considered with preconception and infertility counseling.

Corrigendum: sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance

This corrects the article DOI: 10.1038/nature17392

Abstract A04: Aging microenvironment modulates melanoma invasion and metastasis

The incidence of melanoma rises dramatically after the age of 55. Due to an increase in aging population, it is important to study the change in molecular mechanisms due to aging that would allow development of therapies that are tailored to the age of the patients. The role of the tumor microenvironment in modulating cancer characteristics is widely recognized and it also provides targets for therapeutic intervention. Due to this, we hypothesized that changes in tumor microenvironment due to aging could affect the progression of the melanoma. We obtained skin fibroblasts from healthy donors aged 25-35, as well as skin fibroblasts from healthy donors aged 55-65. We cultured these fibroblasts and used conditioned media from them to affect the invasion of melanoma cells in 3D spheroid invasion assays, where aged fibroblasts promoted invasion of melanoma cells into collagen. We also built artificial skin (reconstructs) using young and aged fibroblasts and demonstrated that skin built with aged fibroblasts promoted melanoma cell invasion. Finally using a transgenic mouse model of melanoma (Yumm1.7, BRAFV600E/PTEN-/-) we observed that the injection of melanoma cells into the tail vein of aged mice (52 weeks) formed metastastic colonies much more rapidly than those injected into the tail vein of young mice (8 weeks). To study the factors involved in the aging microenvironment, we performed a proteomics study of the secretome from young and aged fibroblasts. From this study, we observed that aged fibroblasts secreted inhibitors of canonical Wnt signaling, as well as increased deposition of extracellular matrix components in the aging microenvironment. Since inhibition of canonical Wnt signaling has been linked to decreased sensitivity towards BRAF inhibitors in melanoma, we injected Yumm 1.7 cells subcutaneously into aged and young mice. These mice were then treated with BRAF inhibitors. We observed an increased resistance in response to therapy in the aged mice. We also prepared skin reconstruct from fibroblasts with knockdown of the proteins that we identified from secretome and treated them with PLX4720. These results indicated the role of these extracellular matrix proteins in melanoma. We are exploring the mechanisms of how these extracellular matrix proteins affect the sensitivity towards chemotherapeutics. We concluded that aging could alter tumor microenvironment thus resulting in increased metastasis and therapy resistance. It is important that the studies in cancer therapies take into account the age of the patient to achieve better response in patients. Citation Format: Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Michael O9Connell, Marie Webster, Jessica Appleton, Phil Cheng, Alexander Valiga, Rachel Morissette, Nazli McDonnell, Luigi Ferrucci, Andrew Kossenkov, Katrina Meeth, Marcus Bosenberg, Hsin-Yao Tang, Xiangfan Yin, William Wood, III, Elin Lehrmann, Kevin Becker, Keith Flaherty, Dennie Frederick, Jennifer Wargo, Katherine Aird, Rugang Zhang, Xiaowei Xu, Qin Liu, David Speicher, Ashani Weeraratna. Aging microenvironment modulates melanoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A04.