A Novel Irreversible TEAD Inhibitor, SWTX-143, Blocks Hippo Pathway Transcriptional Output and Causes Tumor Regression in Preclinical Mesothelioma Models.

The Hippo pathway and its downstream effectors, the YAP and TAZ transcriptional coactivators, are deregulated in multiple different types of human cancer and are required for cancer cell phenotypes in vitro and in vivo, while largely dispensable for tissue homeostasis in adult mice. YAP/TAZ and their main partner transcription factors, the TEAD1-4 factors, are therefore promising anticancer targets. Because of frequent YAP/TAZ hyperactivation caused by mutations in the Hippo pathway components NF2 and LATS2, mesothelioma is one of the prime cancer types predicted to be responsive to YAP/TAZ-TEAD inhibitor treatment. Mesothelioma is a devastating disease for which currently no effective treatment options exist. Here, we describe a novel covalent YAP/TAZ-TEAD inhibitor, SWTX-143, that binds to the palmitoylation pocket of all four TEAD isoforms. SWTX-143 caused irreversible and specific inhibition of the transcriptional activity of YAP/TAZ-TEAD in Hippo-mutant tumor cell lines. More importantly, YAP/TAZ-TEAD inhibitor treatment caused strong mesothelioma regression in subcutaneous xenograft models with human cells and in an orthotopic mesothelioma mouse model. Finally, SWTX-143 also selectively impaired the growth of NF2-mutant kidney cancer cell lines, suggesting that the sensitivity of mesothelioma models to these YAP/TAZ-TEAD inhibitors can be extended to other tumor types with aberrations in Hippo signaling. In brief, we describe a novel and specific YAP/TAZ-TEAD inhibitor that has potential to treat multiple Hippo-mutant solid tumor types.

Assessment of Ocriplasmin Effects on the Vitreoretinal Compartment in Porcine and Human Model Systems.

Ocriplasmin (Jetrea®) is a recombinant protease used to treat vitreomacular traction. To gain insight into vitreoretinal observations reported after ocriplasmin treatment, we have developed an in vivo porcine ocriplasmin-induced posterior vitreous detachment (PVD) model in which we investigated vitreoretinal tissues by optical coherence tomography, histology, and cytokine profiling. Eight weeks postinjection, ocriplasmin yielded PVD in 82% of eyes. Subretinal fluid (85%) and vitreous hyperreflective spots (45%) were resolved by week 3. Histological analysis of extracellular matrix (ECM) proteins such as laminin, fibronectin, and collagen IV indicated no retinal ocriplasmin-induced ECM distribution changes. Retinal morphology was unaffected in all eyes. Cytokine profiles of ocriplasmin-treated eyes were not different from vehicle. In cell-based electrical resistance assays, blood-retinal barrier permeability was altered by ocriplasmin concentrations of 6 µg/mL and higher, with all effects being nontoxic, cell-type specific, and reversible. Ocriplasmin was actively taken up by RPE and Müller cells, and our data suggest both lysosomal and transcellular clearance routes for ocriplasmin. In conclusion, transient hyperreflective spots and fluid in a porcine ocriplasmin-induced PVD model did not correlate with retinal ECM rearrangement nor inflammation. Reversible in vitro effects on blood-retinal barrier permeability provide grounds for a hypothesis on the mechanisms behind transient subretinal fluid observed in ocriplasmin-treated patients.

BRCA1 deficiency in skin epidermis leads to selective loss of hair follicle stem cells and their progeny.

The accurate maintenance of genomic integrity is essential for tissue homeostasis. Deregulation of this process leads to cancer and aging. BRCA1 is a critical mediator of this process. Here, we performed conditional deletion of Brca1 during epidermal development and found that BRCA1 is specifically required for hair follicle (HF) formation and for development of adult HF stem cells (SCs). Mice deficient for Brca1 in the epidermis are hairless and display a reduced number of HFs that degenerate progressively. Surprisingly, the interfollicular epidermis and the sebaceous glands remain unaffected by Brca1 deletion. Interestingly, HF matrix transient amplifying progenitors present increased DNA damage, p53 stabilization, and caspase-dependent apoptosis compared with the interfollicular and sebaceous progenitors, leading to hyperproliferation, apoptosis, and subsequent depletion of the prospective adult HF SCs. Concomitant deletion of p53 and Brca1 rescues the defect of HF morphogenesis and loss of HF SCs. During adult homeostasis, BRCA1 is dispensable for quiescent bulge SCs, but upon their activation during HF regeneration, Brca1 deletion causes apoptosis and depletion of Brca1-deficient bulge SCs. Our data reveal a major difference in the requirement of BRCA1 between different types of epidermal SCs and progenitors and during the different activation stages of adult HF SCs.

A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF's ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.

Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death.

Adult stem cells (SCs) are at high risk of accumulating deleterious mutations because they reside and self-renew in adult tissues for extended periods. Little is known about how adult SCs sense and respond to DNA damage within their natural niche. Here, using mouse epidermis as a model, we define the functional consequences and the molecular mechanisms by which adult SCs respond to DNA damage. We show that multipotent hair-follicle-bulge SCs have two important mechanisms for increasing their resistance to DNA-damage-induced cell death: higher expression of the anti-apoptotic gene Bcl-2 and transient stabilization of p53 after DNA damage in bulge SCs. The attenuated p53 activation is the consequence of a faster DNA repair activity, mediated by a higher non-homologous end joining (NHEJ) activity, induced by the key protein DNA-PK. Because NHEJ is an error-prone mechanism, this novel characteristic of adult SCs may have important implications in cancer development and ageing.

Microarray analyses of the effects of NF-kappaB or PI3K pathway inhibitors on the LPS-induced gene expression profile in RAW264.7 cells: synergistic effects of rapamycin on LPS-induced MMP9-overexpression.

Lipopolysaccharide (LPS) activates a broad range of signalling pathways including mainly NF-kappaB and the MAPK cascade, but recent evidence suggests that LPS stimulation also activates the PI3K pathway. To unravel the specific roles of both pathways in LPS signalling and gene expression profiling, we investigated the effects of different inhibitors of NF-kappaB (BAY 11-7082), PI3K (wortmannin and LY294002) but also of mTOR (rapamycin), a kinase acting downstream of PI3K/Akt, in LPS-stimulated RAW264.7 macrophages, analyzing their effects on the LPS-induced gene expression profile using a low density DNA microarray designed to monitor the expression of pro-inflammatory genes. After statistical and hierarchical cluster analyses, we determined five clusters of genes differentially affected by the four inhibitors used. In the fifth cluster corresponding to genes upregulated by LPS and mainly affected by BAY 11-7082, the gene encoding MMP9 displayed a particular expression profile, since rapamycin drastically enhanced the LPS-induced upregulation at both the mRNA and protein levels. Rapamycin also enhanced the LPS-induced NF-kappaB transactivation as determined by a reporter assay, phosphorylation of the p38 and Erk1/2 MAPKs, and counteracted PPAR activity. These results suggest that mTOR could negatively regulate the effects of LPS on the NF-kappaB and MAPK pathways. We also performed real-time RT-PCR assays on mmp9 expression using rosiglitazone (agonist of PPARgamma), PD98059 (inhibitor of Erk 1/2) and SB203580 (inhibitor of p38(MAPK)), that were able to counteract the rapamycin mediated overexpression of mmp9 in response to LPS. Our results suggest a new pathway involving mTOR for regulating specifically mmp9 in LPS-stimulated RAW264.7 cells.

The majority of multipotent epidermal stem cells do not protect their genome by asymmetrical chromosome segregation.

The maintenance of genome integrity in stem cells (SCs) is critical for preventing cancer formation and cellular senescence. The immortal strand hypothesis postulates that SCs protect their genome by keeping the same DNA strand throughout life by asymmetrical cell divisions, thus avoiding accumulation of mutations that can arise during DNA replication. The in vivo relevance of this model remains to date a matter of intense debate. In this study, we revisited this long-standing hypothesis, by analyzing how multipotent hair follicle (HF) SCs segregate their DNA strands during morphogenesis, skin homeostasis, and SC activation. We used three different in vivo approaches to determine how HF SCs segregate their DNA strand during cell divisions. Double-labeling studies using pulse-chase experiments during morphogenesis and the first adult hair cycle showed that HF SCs incorporate two different nucleotide analogs, contradictory to the immortal strand hypothesis. The co-segregation of DNA and chromatin labeling during pulse-chase experiments demonstrated that label retention in HF SCs is rather a mark of relative quiescence. Moreover, DNA labeling of adult SCs, similar to labeling during morphogenesis, also resulted in label retention in HF SCs, indicating that chromosome segregation occurs randomly in most of these cells. Altogether, our results demonstrate that DNA strand segregation occurs randomly in the majority of HF SCs during development, tissue homeostasis, and following SC activation. Disclosure of potential conflicts of interest is found at the end of this article.