Prosocial behavior, social reward and affective state discrimination in adult male and female mice.

Prosocial behavior, defined as voluntary behavior intended to benefit another, has long been regarded as a primarily human characteristic. In recent years, it was reported that laboratory animals also favor prosocial choices in various experimental paradigms, thus demonstrating that prosocial behaviors are evolutionarily conserved. Here, we investigated prosocial choices in adult male and female C57BL/6 laboratory mice in a task where a subject mouse was equally rewarded for entering any of the two compartments of the experimental cage, but only entering of the compartment designated as "prosocial" rewarded an interaction partner. In parallel we have also assessed two traits that are regarded as closely related to prosociality: sensitivity to social reward and the ability to recognize the affective state of another individual. We found that female, but not male, mice increased frequency of prosocial choices from pretest to test. However, both sexes showed similar rewarding effects of social contact in the conditioned place preference test, and similarly, there was no effect of sex on affective state discrimination measured as the preference for interaction with a hungry or relieved mouse over a neutral animal. These observations bring interesting parallels to differences between sexes observed in humans, and are in line with reported higher propensity for prosocial behavior in human females, but differ with regard to sensitivity to social stimuli in males.

C57BL/6N mice show a sub-strain specific resistance to the psychotomimetic effects of ketamine.

Repeated administration of subanesthetic doses of ketamine is a model of psychosis-like state in rodents. In mice, this treatment produces a range of behavioral deficits, including impairment in social interactions and locomotion. To date, these phenotypes were described primarily in the Swiss and C3H/HeHsd mouse strains. A few studies investigated ketamine-induced behaviors in the C57BL/6J strain, but to our knowledge the C57BL/6N strain was not investigated thus far. This is surprising, as both C57BL/6 sub-strains are widely used in behavioral and neuropsychopharmacological research, and are de facto standards for characterization of drug effects. The goal of this study was to determine if C57BL/6N mice are vulnerable to develop social deficits after 5 days withdrawal from sub-chronic ketamine treatment (5 days, 30 mg/kg, i.p.), an experimental schedule shown before to cause deficits in social interactions in C57BL/6J mice. Our results show that sub-chronic administration of ketamine that was reported to cause psychotic-like behavior in C57BL/6J mice does not induce appreciable behavioral alterations in C57BL/6N mice. Thus, we show that the effects of sub-chronic ketamine treatment in mice are sub-strain specific.

Establishment of a social conditioned place preference paradigm for the study of social reward in female mice.

Social interactions can be and often are rewarding. The effect of social contact strongly depends on circumstances, and the reward may be driven by varied motivational processes, ranging from parental or affiliative behaviors to investigation or aggression. Reward associated with nonreproductive interactions in rodents is measured using the social conditioned place preference (sCPP) paradigm, where a change in preference for an initially neutral context confirms reinforcing effects of social contact. Here, we revised the sCPP method and reexamined social reward in adult female mice. Contrary to earlier studies, we found that robust rewarding effects of social contact could be detected in adult (14-week-old) female C57BL/6 mice when the sCPP task was refined to remove confounding factors. Strikingly, the rewarding effects of social interaction were only observed among female siblings who remained together from birth. Contact with same-age nonsiblings was not rewarding even after 8 weeks of cohousing. Other factors critical for the social reward effect in the sCPP paradigm included the number of conditioning sessions and the inherent preference for contextual cues. Thus, we show that social interaction is rewarding in adult female mice, but this effect strictly depends on the familiarity of the interaction partners. Furthermore, by identifying confounding factors, we provide a behavioral model to study the mechanisms underlying the rewarding effects of nonreproductive social interaction in adult mice.

Disruption of RING and PHD Domains of TRIM28 Evokes Differentiation in Human iPSCs.

TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells' (ESC) self-renewal potential. As the epigenetic factor modulating chromatin structure, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. Because of many similarities between highly dedifferentiated cancer cells and normal pluripotent stem cells, we applied human induced pluripotent stem cells (hiPSC) as a model for stemness studies. For the first time in hiPSC, we analyzed the function of individual TRIM28 domains. Here we demonstrate the essential role of a really interesting new gene (RING) domain and plant homeodomain (PHD) in regulating pluripotency maintenance and self-renewal capacity of hiPSC. Our data indicate that mutation within the RING or PHD domain leads to the loss of stem cell phenotypes and downregulation of the FGF signaling. Moreover, impairment of RING or PHD domain results in decreased proliferation and impedes embryoid body formation. In opposition to previous data indicating the impact of phosphorylation on TRIM28 function, our data suggest that TRIM28 phosphorylation does not significantly affect the pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with disrupted RING and PHD functions display downregulation of genes associated with tumor metastasis, which are considered important targets in cancer treatment. Our data suggest the potential use of RING and PHD domains of TRIM28 as targets in cancer therapy.

Loss of mu and delta opioid receptors on neurons expressing dopamine receptor D1 has no effect on reward sensitivity.

Opioid signaling controls the activity of the brain's reward system. It is involved in signaling the hedonic effects of rewards and has essential roles in reinforcement and motivational processes. Here, we focused on opioid signaling through mu and delta receptors on dopaminoceptive neurons and evaluated the role these receptors play in reward-driven behaviors. We generated a genetically modified mouse with selective double knockdown of mu and delta opioid receptors in neurons expressing dopamine receptor D1. Selective expression of the transgene was confirmed using immunostaining. Knockdown was validated by measuring the effects of selective opioid receptor agonists on neuronal membrane currents using whole-cell patch clamp recordings. We found that in the nucleus accumbens of control mice, the majority of dopamine receptor D1-expressing neurons were sensitive to a mu or delta opioid agonist. In mutant mice, the response to the delta receptor agonist was blocked, while the effects of the mu agonist were strongly attenuated. Behaviorally, the mice had no obvious impairments. The mutation did not affect the sensitivity to the rewarding effects of morphine injections or social contact and had no effect on preference for sweet taste. Knockdown had a moderate effect on motor activity in some of the tests performed, but this effect did not reach statistical significance. Thus, we found that knocking down mu and delta receptors on dopamine receptor D1-expressing cells does not appreciably affect some of the reward-driven behaviors previously attributed to opioid signaling.

HSPA2 Chaperone Contributes to the Maintenance of Epithelial Phenotype of Human Bronchial Epithelial Cells but Has Non-Essential Role in Supporting Malignant Features of Non-Small Cell Lung Carcinoma, MCF7, and HeLa Cancer Cells.

Heat Shock Protein A2 (HSPA2) is a member of the HSPA (HSP70) chaperone family and has a critical role for male fertility. HSPA2 is present in a number of somatic organs. Limited evidence suggests that HSPA2 may be involved in regulating epithelial cell differentiation. HSPA2 also emerged as a cancer-related chaperone; however, no consensus on its functional significance has been reached so far. In this study, we compared the phenotypic effects of HSPA2 deficit in non-transformed human bronchial epithelial cells (HBEC), and in lung, breast, and cervical cancer cells. We used various techniques to inhibit the HSPA2 gene expression in order to examine the impact of HSPA2 deficiency on cell growth, migration, adhesion, and invasion. Our results show that HBEC but not cancer cells are sensitive to HSPA2 deficit. HSPA2 knockdown in HBEC cells impaired their clone-forming ability and adhesiveness. Thus, our results indicate that epithelial cells can rely on a specific activity of HSPA2, but such dependence can be lost in epithelial cells that have undergone malignant transformation.

Heat shock proteins create a signature to predict the clinical outcome in breast cancer.

Utilizing The Cancer Genome Atlas (TCGA) and KM plotter databases we identified six heat shock proteins associated with survival of breast cancer patients. The survival curves of samples with high and low expression of heat shock genes were compared by log-rank test (Mantel-Haenszel). Interestingly, patients overexpressing two identified HSPs - HSPA2 and DNAJC20 exhibited longer survival, whereas overexpression of other four HSPs - HSP90AA1, CCT1, CCT2, CCT6A resulted in unfavorable prognosis for breast cancer patients. We explored correlations between expression level of HSPs and clinicopathological features including tumor grade, tumor size, number of lymph nodes involved and hormone receptor status. Additionally, we identified a novel signature with the potential to serve as a prognostic model for breast cancer. Using univariate Cox regression analysis followed by multivariate Cox regression analysis, we built a risk score formula comprising prognostic HSPs (HSPA2, DNAJC20, HSP90AA1, CCT1, CCT2) and tumor stage to identify high-risk and low-risk cases. Finally, we analyzed the association of six prognostic HSP expression with survival of patients suffering from other types of cancer than breast cancer. We revealed that depending on cancer type, each of the six analyzed HSPs can act both as a positive, as well as a negative regulator of cancer development. Our study demonstrates a novel HSP signature for the outcome prediction of breast cancer patients and provides a new insight into ambiguous role of these proteins in cancer development.

Molecular chaperones in the acquisition of cancer cell chemoresistance with mutated TP53 and MDM2 up-regulation.

Utilizing the TCGA PANCAN12 dataset we discovered that cancer patients with mutations in TP53 tumor suppressor and overexpression of MDM2 oncogene exhibited decreased survival post treatment. Interestingly, in the case of breast cancer patients, this phenomenon correlated with high expression level of several molecular chaperones belonging to the HSPA, DNAJB and HSPC families. To verify the hypothesis that such a genetic background may promote chaperone-mediated chemoresistance, we employed breast and lung cancer cell lines that constitutively overexpressed heat shock proteins and have shown that HSPA1A/HSP70 and DNAJB1/HSP40 facilitated the binding of mutated p53 to the TAp73α protein. This chaperone-mediated mutated p53-TAp73α complex induced chemoresistance to DNA damaging reagents, like Cisplatin, Doxorubicin, Etoposide or Camptothecin. Importantly, when the MDM2 oncogene was overexpressed, heat shock proteins were displaced and a stable multiprotein complex comprising of mutated p53-TAp73α-MDM2 was formed, additionally amplifying cancer cells chemoresistance. Our findings demonstrate that molecular chaperones aid cancer cells in surviving the cytotoxic effect of chemotherapeutics and may have therapeutic implications.

TRIM28 epigenetic corepressor is indispensable for stable induced pluripotent stem cell formation.

Cellular reprogramming proceeds in a stepwise pathway initiated by binding and transcription of pluripotency factors followed by genome-wide epigenetic changes. Priming events, such as erasure of DNA methylation and chromatin remodeling determines the success of pluripotency acquisition later. Therefore, growing efforts are made to understand epigenetic regulatory network that makes reprogramming possible and efficient. Here, we analyze the role of transcriptional corepressor TRIM28, involved in heterochromatin formation, during the process of reprogramming of mouse somatic cells into induced pluripotent stem cells (iPS cells). We demonstrate that Trim28 knockdown (Trim28 KD) causes that emerging iPS cells differentiate immediately back into MEFs therefore they fail to yield stable iPS cell colonies. To better comprehend the mechanism of TRIM28 action in reprogramming, we performed a reverse-phase protein array (RPPA) using in excess of 300 different antibodies and compared the proteomic profiles of wild-type and Trim28 KD cells during reprogramming. We revealed the differences in the dynamics of reprogramming of wild-type and Trim28 KD cells. Interestingly, proteomic profile of Trim28 KD cells at the final stage of reprogramming resembled differentiated state rather than maintenance of pluripotency and self-renewal, strongly suggesting spontaneous differentiation of Trim28 KD cells back to their parental cell type. We also observed that action of TRIM28 in reprogramming is accompanied by differential enrichment of proteins involved in cell cycle, adhesion and stemness. Collectively, these results suggest that regulation of epigenetic modifications coordinated by TRIM28 plays a crucial role in reprogramming process.

TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated the role of TRIM28 protein in the regulation of breast cancer stem cells (CSC) populations and tumorigenesis in vitro and in vivo. Downregulation of TRIM28 expression in xenografts led to deceased expression of pluripotency and mesenchymal markers, as well as inhibition of signaling pathways involved in the complex mechanism of CSC maintenance. Moreover, TRIM28 depletion reduced the ability of cancer cells to induce tumor growth when subcutaneously injected in limiting dilutions. Our data demonstrate that the downregulation of TRIM28 gene expression reduced the ability of CSCs to self-renew that resulted in significant reduction of tumor growth. Loss of function of TRIM28 leads to dysregulation of cell cycle, cellular response to stress, cancer cell metabolism, and inhibition of oxidative phosphorylation. All these mechanisms directly regulate maintenance of CSC population. Our original results revealed the role of the TRIM28 in regulating the CSC population in breast cancer. These findings may pave the way to novel and more effective therapies targeting cancer stem cells in breast tumors.

Oncogenesis and induced pluripotency - commonalities of signalling pathways.

Rapid progress in the field of adult cells reprogramming back into a stem cell-like fate revealed shared mechanisms of action with tumoural reprogramming. A hallmark of stem cells - self-renewal and differentiation potential - seems to be tightly interlaced with large proliferation capacity and cellular plasticity of cancer cells. In this review, we briefly summarise the core transcription factors critical to maintenance of ES cell signature and overexpressed in many types of cancer, as well as signalling pathways involved in both induced pluripotency and oncogenesis, with particular regard to the role of tumour suppressor p53.