Exploring Adaptive Phenotypes for the Human Calcium-Sensing Receptor Polymorphism R990G.

Rainforest hunter-gatherers from Southeast Asia are characterized by specific morphological features including a particularly dark skin color (D), short stature (S), woolly hair (W), and the presence of steatopygia (S)-fat accumulation localized in the hips (DSWS phenotype). Based on previous evidence in the Andamanese population, we first characterized signatures of adaptive natural selection around the calcium-sensing receptor gene in Southeast Asian rainforest groups presenting the DSWS phenotype and identified the R990G substitution (rs1042636) as a putative adaptive variant for experimental follow-up. Although the calcium-sensing receptor has a critical role in calcium homeostasis by directly regulating the parathyroid hormone secretion, it is expressed in different tissues and has been described to be involved in many biological functions. Previous works have also characterized the R990G substitution as an activating polymorphism of the calcium-sensing receptor associated with hypocalcemia. Therefore, we generated a knock-in mouse for this substitution and investigated organismal phenotypes that could have become adaptive in rainforest hunter-gatherers from Southeast Asia. Interestingly, we found that mouse homozygous for the derived allele show not only lower serum calcium concentration but also greater body weight and fat accumulation, probably because of enhanced preadipocyte differentiation and lipolysis impairment resulting from the calcium-sensing receptor activation mediated by R990G. We speculate that such differential features in humans could have facilitated the survival of hunter-gatherer groups during periods of nutritional stress in the challenging conditions of the Southeast Asian tropical rainforests.

Distinct roles for PARP-1 and PARP-2 in c-Myc-driven B-cell lymphoma in mice.

Dysregulation of the c-Myc oncogene occurs in a wide variety of hematologic malignancies, and its overexpression has been linked with aggressive tumor progression. Here, we show that poly (ADP-ribose) polymerase 1 (PARP-1) and PARP-2 exert opposing influences on progression of c-Myc-driven B-cell lymphoma. PARP-1 and PARP-2 catalyze the synthesis and transfer of ADP-ribose units onto amino acid residues of acceptor proteins in response to DNA strand breaks, playing a central role in the response to DNA damage. Accordingly, PARP inhibitors have emerged as promising new cancer therapeutics. However, the inhibitors currently available for clinical use are not able to discriminate between individual PARP proteins. We found that genetic deletion of PARP-2 prevents c-Myc-driven B-cell lymphoma, whereas PARP-1 deficiency accelerates lymphomagenesis in the Eµ-Myc mouse model of aggressive B-cell lymphoma. Loss of PARP-2 aggravates replication stress in preleukemic Eµ-Myc B cells, resulting in accumulation of DNA damage and concomitant cell death that restricts the c-Myc-driven expansion of B cells, thereby providing protection against B-cell lymphoma. In contrast, PARP-1 deficiency induces a proinflammatory response and an increase in regulatory T cells, likely contributing to immune escape of B-cell lymphoma, resulting in an acceleration of lymphomagenesis. These findings pinpoint specific functions for PARP-1 and PARP-2 in c-Myc-driven lymphomagenesis with antagonistic consequences that may help inform the design of new PARP-centered therapeutic strategies, with selective PARP-2 inhibition potentially representing a new therapeutic approach for the treatment of c-Myc-driven tumors.

Coordinated signals from PARP-1 and PARP-2 are required to establish a proper T cell immune response to breast tumors in mice.

Poly(ADP-ribose)-polymerase (PARP)-1 and PARP-2 play an essential role in the DNA damage response. Based on this effect of PARP in the tumor cell itself, PARP inhibitors have emerged as new therapeutic tools both approved and in clinical trials. However, the interactome of multiple other cell types, particularly T cells, within the tumor microenvironment are known to either favor or limit tumorigenesis. Here, we bypassed the embryonic lethality of dually PARP-1/PARP-2-deficient mice by using a PARP-1-deficient mouse with a Cd4-promoter-driven deletion of PARP-2 in T cells to investigate the understudied role of these PARPs in the modulation of T cell responses against AT-3-induced breast tumors. We found that dual PARP-1/PARP-2-deficiency in T cells promotes tumor growth while single deficiency of each protein limited tumor progression. Analysis of tumor-infiltrating cells in dual PARP-1/PARP-2-deficiency host-mice revealed a global change in immunological profile and impaired recruitment and activation of T cells. Conversely, single PARP-1 and PARP-2-deficiency tends to produce an environment with an active and partially upregulated immune response. Our findings pinpoint opposite effects of single and dual PARP-1 and PARP-2-deficiency in modulating the antitumor response with an impact on tumor progression, and will have implications for the development of more selective PARP-centered therapies.

Coordinated signals from the DNA repair enzymes PARP-1 and PARP-2 promotes B-cell development and function.

Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 regulate the function of various DNA-interacting proteins by transferring ADP-ribose emerging from catalytic cleavage of cellular ß-NAD+. Hence, mice lacking PARP-1 or PARP-2 show DNA perturbations ranging from altered DNA integrity to impaired DNA repair. These effects stem from the central role that PARP-1 and PARP-2 have on the cellular response to DNA damage. Failure to mount a proper response culminates in cell death. Accordingly, PARP inhibitors are emerging as promising drugs in cancer therapy. However, the full impact of these inhibitors on immunity, including B-cell antibody production, remains elusive. Given that mice carrying dual PARP-1 and PARP-2 deficiency develop early embryonic lethality, we crossed PARP-1-deficient mice with mice carrying a B-cell-conditional PARP-2 gene deletion. We found that the resulting dually PARP-1 and PARP-2-deficient mice had perturbed bone-marrow B-cell development as well as profound peripheral depletion of transitional and follicular but not marginal zone B-cells. Of note, bone-marrow B-cell progenitors and peripheral mature B-cells were conserved in mice carrying either PARP-1 or PARP-2 deficiency. In dually PARP-1 and PARP-2-deficient mice, B-cell lymphopenia was associated with increased DNA damage and accentuated death in actively proliferating B-cells. Moreover, dual PARP-1 and PARP-2 deficiency impaired antibody responses to T-independent carbohydrate but not to T-dependent protein antigens. Notwithstanding the pivotal role of PARP-1 and PARP-2 in DNA repair, combined PARP-1 and PARP-2 deficiency did not perturb the DNA-editing processes required for the generation of a protective antibody repertoire, including Ig V(D)J gene recombination and IgM-to-IgG class switching. These findings provide key information as to the potential impact of PARP inhibitors on humoral immunity, which will facilitate the development of safer PARP-targeting regimens against cancer.

Identity Noise and Adipogenic Traits Characterize Dermal Fibroblast Aging.

During aging, stromal functions are thought to be impaired, but little is known whether this stems from changes of fibroblasts. Using population- and single-cell transcriptomics, as well as long-term lineage tracing, we studied whether murine dermal fibroblasts are altered during physiological aging under different dietary regimes that affect longevity. We show that the identity of old fibroblasts becomes undefined, with the fibroblast states present in young skin no longer clearly demarcated. In addition, old fibroblasts not only reduce the expression of genes involved in the formation of the extracellular matrix, but also gain adipogenic traits, paradoxically becoming more similar to neonatal pro-adipogenic fibroblasts. These alterations are sensitive to systemic metabolic changes: long-term caloric restriction reversibly prevents them, whereas a high-fat diet potentiates them. Our results therefore highlight loss of cell identity and the acquisition of adipogenic traits as a mechanism underlying cellular aging, which is influenced by systemic metabolism.

Macrophage-specific MHCII expression is regulated by a remote Ciita enhancer controlled by NFAT5.

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

PARP-1/PARP-2 double deficiency in mouse T cells results in faulty immune responses and T lymphomas.

The maintenance of T-cell homeostasis must be tightly regulated. Here, we have identified a coordinated role of Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 in maintaining T-lymphocyte number and function. Mice bearing a T-cell specific deficiency of PARP-2 in a PARP-1-deficient background showed defective thymocyte maturation and diminished numbers of peripheral CD4+ and CD8+ T-cells. Meanwhile, peripheral T-cell number was not affected in single PARP-1 or PARP-2-deficient mice. T-cell lymphopenia was associated with dampened in vivo immune responses to synthetic T-dependent antigens and virus, increased DNA damage and T-cell death. Moreover, double-deficiency in PARP-1/PARP-2 in T-cells led to highly aggressive T-cell lymphomas with long latency. Our findings establish a coordinated role of PARP-1 and PARP-2 in T-cell homeostasis that might impact on the development of PARP-centred therapies.

miRNA-1 and miRNA-133a are involved in early commitment of pluripotent stem cells and demonstrate antagonistic roles in the regulation of cardiac differentiation.

miRNA-1 (miR-1) and miRNA-133a (miR-133a) are muscle-specific miRNAs that play an important role in heart development and physiopathology. Although both miRNAs have been broadly studied during cardiogenesis, the mechanisms by which miR-1 and miR-133a could influence linage commitment in pluripotent stem cells remain poorly characterized. In this study we analysed the regulation of miR-1 and miR-133a expression during pluripotent stem cell differentiation [P19.CL6 cells; embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and investigated their role in DMSO and embryoid body (EB)-mediated mesodermal and cardiac differentiation by gain- and loss-of-function studies, as well as in vivo, by the induction of teratomas. Gene expression analysis revealed that miR-1 and miR-133a are upregulated during cardiac differentiation of P19.CL6 cells, and also during ESC and iPSC EB differentiation. Forced overexpression of both miRNAs promoted mesodermal commitment and a concomitant decrease in the expression of neural differentiation markers. Moreover, overexpression of miR-1 enhanced the cardiac differentiation of P19.CL6, while miR-133a reduced it with respect to control cells. Teratoma formation experiments with P19.CL6 cells confirmed the influence of miR-1 and miR-133a during in vivo differentiation. Finally, inhibition of both miRNAs during P19.CL6 cardiac differentiation had opposite results to their overexpression. In conclusion, gene regulation involving miR-1 and miR-133a controls the mesodermal and cardiac fate of pluripotent stem cells. Copyright © 2014 John Wiley & Sons, Ltd.

A cellular model reflecting the phenotypic heterogeneity of mutant HRAS driven squamous cell carcinoma.

Squamous cell carcinomas have a range of histopathological manifestations. The parameters that determine this clinically observed heterogeneity are not fully understood. Here, we report the generation of a cell culture model that reflects part of this heterogeneity. We have used the catalytic subunit of human telomerase hTERT and large T to immortalize primary UV-unexposed keratinocytes. Then, mutant HRAS G12V has been introduced to transform these immortal keratinocytes. When injected into immunosuppressed mice, transformed cells grew as xenografts with distinct histopathological characteristics. We observed three major tissue architectures: solid, sarcomatoid and cystic growth types, which were primarily composed of pleomorphic and basaloid cells but in some cases displayed focal apocrine differentiation. We demonstrate that the cells generated represent different stages of skin cancerogenesis and as such can be used to identify novel tumor-promoting alterations such as the overexpression of the PADI2 oncogene in solid-type SCC. Importantly, the cultured cells maintain the characteristics from the xenograft they were derived from while being amenable to manipulation and analysis. The availability of cell lines representing different clinical manifestations opens a new tool to study the stochastic and deterministic factors that cause case-to-case heterogeneity despite departing from the same set of oncogenes and the same genetic background.

A mouse model uncovers LKB1 as an UVB-induced DNA damage sensor mediating CDKN1A (p21WAF1/CIP1) degradation.

Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.

The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells.

In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfß-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy.

Lkb1 loss promotes tumor progression of BRAF(V600E)-induced lung adenomas.

Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAF(V600E)) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAF(V600E)-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAF(V600E) oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAF(V600E) leads to lung adenoma development. Importantly, expression of BRAF(V600E) concomitant with the loss of only a single-copy of Lkb1, overcomes senencence-like features of BRAF(V600E)-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAF(V600E) mutated lung adenomas in human cancer patients.

Parp-2 is required to maintain hematopoiesis following sublethal γ-irradiation in mice.

Hematopoietic stem cells self-renew for life to guarantee the continuous supply of all blood cell lineages. Here we show that Poly(ADP-ribose) polymerase-2 (Parp-2) plays an essential role in hematopoietic stem/progenitor cells (HSPC) survival under steady-state conditions and in response to stress. Increased levels of cell death were observed in HSPC from untreated Parp-2-/- mice, but this deficit was compensated by increased rates of self-renewal, associated with impaired reconstitution of hematopoiesis upon serial bone marrow transplantation. Cell death after γ-irradiation correlated with an impaired capacity to repair DNA damage in the absence of Parp-2. Upon exposure to sublethal doses of γ-irradiation, Parp-2-/- mice exhibited bone marrow failure that correlated with reduced long-term repopulation potential of irradiated Parp-2-/- HSPC under competitive conditions. In line with a protective role of Parp-2 against irradiation-induced apoptosis, loss of p53 or the pro-apoptotic BH3-only protein Puma restored survival of irradiated Parp-2-/- mice, whereas loss of Noxa had no such effect. Our results show that Parp-2 plays essential roles in the surveillance of genome integrity of HSPC by orchestrating DNA repair and restraining p53-induced and Puma-mediated apoptosis. The data may affect the design of drugs targeting Parp proteins and the improvement of radiotherapy-based therapeutic strategies.

Staphylococcus prevails in the skin microbiota of long-term immunodeficient mice.

Host-commensal relationships in the skin are a complex system governed by variables related to the host, the bacteria and the environment. A disruption of this system may lead to new steady states, which, in turn, may lead to disease. We have studied one such disruption by characterizing the skin microbiota in healthy and immunodepressed (ID) mice. A detailed anatomopathological study failed to reveal any difference between the skin of healthy and ID mice. We sequenced the 16S rDNA V1-V2 gene region to saturation in 10 healthy and 10 ID 8 week-old mice, and found than all of the healthy and two of the ID mice had bacterial communities that were similar in composition to that of human skin, although, presumably because of the uniform raising conditions, less interindividual variation was found in mice. However, eight ID mice showed microbiota dominated by Staphylococcus epidermidis. Quantitative PCR amplification of 16S rDNA gene and of the Staphylococcus-specific TstaG region confirmed the previous results and indicated that the quantitative levels of Staphylococcus were similar in both groups while the total number of 16S copies was greater in the healthy mice. Thus, it is possible that, under long-term immunodeficiency, which removes the acquired but not the native immune system, S.epidermidis may inhibit the growth of other bacteria but does not cause a pathogenic state.

MacroH2A1 regulates the balance between self-renewal and differentiation commitment in embryonic and adult stem cells.

One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.

Chimeric infectious bursal disease virus-like particles as potent vaccines for eradication of established HPV-16 E7-dependent tumors.

Cervical cancer is caused by persistent high-risk human papillomavirus (HR-HPV) infection and represents the second most frequent gynecological malignancy in the world. The HPV-16 type accounts for up to 55% of all cervical cancers. The HPV-16 oncoproteins E6 and E7 are necessary for induction and maintenance of malignant transformation and represent tumor-specific antigens for targeted cytotoxic T lymphocyte-mediated immunotherapy. Therapeutic cancer vaccines have become a challenging area of oncology research in recent decades. Among current cancer immunotherapy strategies, virus-like particle (VLP)-based vaccines have emerged as a potent and safe approach. We generated a vaccine (VLP-E7) incorporating a long C-terminal fragment of HPV-16 E7 protein into the infectious bursal disease virus VLP and tested its therapeutic potential in HLA-A2 humanized transgenic mice grafted with TC1/A2 tumor cells. We performed a series of tumor challenge experiments demonstrating a strong immune response against already-formed tumors (complete eradication). Remarkably, therapeutic efficacy was obtained with a single dose without adjuvant and against two injections of tumor cells, indicating a potent and long-lasting immune response.

E-box-independent regulation of transcription and differentiation by MYC.

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.

PARP-1 and PARP-2: New players in tumour development.

Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 belong to a family of enzymes that, using NAD(+) as a substrate, catalyze poly(ADP-ribosyl)ation of proteins. PARP-1 and PARP-2 catalytic activity is stimulated by DNA-strand breaks targeting mainly proteins involved in chromatin structure and DNA metabolism, providing strong support for a dual role of both PARP-1 and PARP-2 in the DNA damage response as DNA damage sensors and signal transducers to downstream effectors. The DNA damage response has important consequences for genomic stability and tumour development. In order to manipulate DNA damage responses to selectively induce tumour cell death, a considerable effort is centred on defining the molecular mechanisms that allow cells to detect, respond to, and repair DNA damage. PARP inhibitors that compete with NAD+ at the highly conserved enzyme active site are arisen as new potential therapeutic strategies as chemo- and radiopotentiation and for the treatment of cancers with specific DNA repair defects as single-agent therapies. In the present review, we highlight emerging information about the redundant and specific functions of PARP-1 and PARP-2 in genome surveillance and DNA repair pathways. Understanding these roles might provide invaluable clues to design new cancer therapeutic approaches. In addition, we provide an overview of ongoing clinical trials with PARP inhibitors and the value of PARP-1 and PARP-2 expression as prognostic biomarkers in cancer.