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MicroRNAs (miR) are small RNA molecules that shape the cell transcriptome and proteome through regulation of mRNA stability and translation. Here, we examined their function as determinants of cell resistance to complement-dependent cytotoxicity (CDC). To achieve this goal, we compared the expression of microRNAs between complement-resistant and -sensitive K562 leukemia, Raji lymphoma, and HCT-116 colorectal carcinoma cells. Global microRNA array analysis identified miR-150, miR-328, and miR-616 as regulators of CDC resistance. Inhibition of miR-150 reduced resistance, whereas inhibition of miR-328 or miR-616 enhanced cell resistance. Treatment of K562 cells with a sublytic dose of complement was shown to rapidly increase miR-150, miR-328, and miR-616 expression. Protein targets of these microRNAs were analyzed in K562 cells by mass spectrometry–based proteomics. Expression of the complement membrane regulatory proteins CD46 and CD59 was significantly enhanced after inhibition of miR-328 and miR-616. Enrichment of proteins of mitochondria, known target organelles in CDC, was observed after miR-150, miR-328, and miR-616 inhibition. In conclusion, miR-150, miR-328, and miR-616 regulate cell resistance to CDC by modifying the expression of the membrane complement regulators CD46 and CD59 and the response of the mitochondria to complement lytic attack. These microRNAs may be considered targets for intervention in complement-associated diseases and in anticancer, complement-based therapy.

SummaryDespite extensive research, internal tumor heterogeneity presents enormous challenges to achieve complete therapeutic responses. Changes in protein expression are central determinants of cancer phenotypes that reflect potential therapeutic targets. However, previous proteomic studies did not address internal heterogeneity, therefore, masked the necessary spatial resolution to achieve a comprehensive understanding of cancer complexity. Here we present the first large-scale multi-focal breast cancer proteomic study of 330 tumor regions which associated cancer cell function, pathological parameters, and spatial localization of each tumor region. We found marked internal proteomic heterogeneity even within tumors presenting homogeneous receptor expression. Additionally, analysis of the internal heterogeneity, based on coexisting receptor expression or histological patterns in single tumors, showed significant functional differences between homogeneous and heterogeneous tumors related to cancer metabolism, immunogenicity, and proliferation. We anticipate that this study will serve as a starting point towards the development of improved cancer therapy and diagnostics.

Summary Ultraviolet (UV) light affects endocrinological and behavioral aspects of sexuality via an unknown mechanism. Here we discover that ultraviolet B (UVB) exposure enhances the levels of sex-steroid hormones and sexual behavior, which are mediated by the skin. In female mice, UVB exposure increases hypothalamus-pituitary-gonadal axis hormone levels, resulting in larger ovaries; extends estrus days; and increases anti-Mullerian hormone (AMH) expression. UVB exposure also enhances the sexual responsiveness and attractiveness of females and male-female interactions. Conditional knockout of p53 specifically in skin keratinocytes abolishes the effects of UVB. Thus, UVB triggers a skin-brain-gonadal axis through skin p53 activation. In humans, solar exposure enhances romantic passion in both genders and aggressiveness in men, as seen in analysis of individual questionaries, and positively correlates with testosterone level. Our findings suggest opportunities for treatment of sex-steroid-related dysfunctions., Graphical abstract, Highlights • UVB exposure increases circulating sex-steroid levels in mice and humans • UVB exposure enhances female attractiveness and receptiveness toward males • UVB exposure increases females’ estrus phase, HPG axis hormones, and follicle growth • Skin p53 regulates UVB-induced sexual behavior and ovarian physiological changes, Parikh et al. find that UVB exposure triggers a skin-brain-gonadal axis through skin p53 activation. UVB exposure increases in female mice sexual responsiveness and attractiveness, hypothalamus-pituitary-gonadal axis hormone levels, ovary size, and estrus duration, as well as male-female interactions. Solar exposure in humans enhances romantic passion and positively correlates with male testosterone levels.

Sexual dimorphisms are responsible for profound metabolic differences in health and behavior. Whether males and females react differently to environmental cues, such as solar ultraviolet (UV) exposure, is unknown. Here we show that solar exposure induces food-seeking behavior, food intake, and food-seeking behavior and food intake in men, but not in women, through epidemiological evidence of approximately 3,000 individuals throughout the year. In mice, UVB exposure leads to increased food-seeking behavior, food intake and weight gain, with a sexual dimorphism towards males. In both mice and human males, increased appetite is correlated with elevated levels of circulating ghrelin. Specifically, UVB irradiation leads to p53 transcriptional activation of ghrelin in skin adipocytes, while a conditional p53-knockout in mice abolishes UVB-induced ghrelin expression and food-seeking behavior. In females, estrogen interferes with the p53–chromatin interaction on the ghrelin promoter, thus blocking ghrelin and food-seeking behavior in response to UVB exposure. These results identify the skin as a major mediator of energy homeostasis and may lead to therapeutic opportunities for sex-based treatments of endocrine-related diseases.

Summary Glioblastoma (GBM) is the most aggressive form of glioma, with poor prognosis exhibited by most patients, and a median survival time of less than 2 years. We assemble a cohort of 87 GBM patients whose survival ranges from less than 3 months and up to 10 years and perform both high-resolution mass spectrometry proteomics and RNA sequencing (RNA-seq). Integrative analysis of protein expression, RNA expression, and patient clinical information enables us to identify specific immune, metabolic, and developmental processes associated with survival as well as determine whether they are shared between expression layers or are layer specific. Our analyses reveal a stronger association between proteomic profiles and survival and identify unique protein-based classification, distinct from the established RNA-based classification. By integrating published single-cell RNA-seq data, we find a connection between subpopulations of GBM tumors and survival. Overall, our findings establish proteomic heterogeneity in GBM as a gateway to understanding poor survival.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations. Straussman and colleagues undertake clonal analyses and show that drug tolerance to EGFR therapy in lung cancer cell populations is an inherited continuous trait that is determined by IRS1 phosphorylation.

Ultraviolet (UV) light affects endocrinological and behavioral aspects of human sexuality via an unknown mechanism. Using a unique male-female comparative approach, we discovered that the sexual behavioral and hormonal features enhanced by UVB are mediated by the skin. In mice, UV exposure increases hypothalamus-pituitary-gonadal axis hormone levels, resulting in enhanced ovary size, extended estrus days, and anti-Mullerian hormone (AMH) expression. It likewise enhances sexual responsiveness and attractiveness of females and heterosexual interactions of both males and females. Conditional knockout of p53 specifically in skin keratinocytes abolished UV’s effects. In humans, UV exposure enhanced romantic passion in both genders, increased testosterone levels in men and correlated with heightened plasma AMH levels in women. Our data, revealing that UVB triggers a skin-brain-gonadal axis through skin p53 activation, offers therapeutic opportunities for sex-steroid-related dysfunctions. We speculate that during human furless skin evolution, the skin became the front-line regulator of the response to UVB.

Obesity has become one of the leading pathophysiologic disorders in recent years. Adipose tissue is the main tissue related to obesity and is known to play a role in various physiological complications, including type 2 diabetes. To better understand how the fat tissue develops, we used an in vitro live cell imaging system to quantify the adipogenesis by means of nondestructive digital imaging to monitor the accumulation of intracellular lipid droplets (LDs), a hallmark of adipogenesis, from the macro- to the micro-scale. Analyzing the cells' shape at the single-cell level allows to quantify the cells' shape change from a fibroblast to spherical morphology, indicating the start of adipogenesis. To reveal the molecular alterations, we applied a proteomic approach using high-resolution mass spectrometry of the proliferation, confluent fibroblasts and of adipocytes. During this process, we noted the reorganization of the cells' extracellular matrix (ECM) network microenvironment from fibrillary collagen types I, III and V to collagens IV and VI, which affected the cells niche. The changes in ECM are translated for cytoskeleton remodeling according to cell fate-determining mechanisms. We quantified the cytoskeleton rearrangement of long oriented actin fibers or short cortical and disorganized fibers, associated with LDs accumulation in adipocytes. Developing in vitro models and analytical methods enable us to study differentiation into adipocytes that will advance our understanding regarding the niche conditions that affect adipogenesis. Consequently, this will enable the development of new modalities to prevent obesity and its deleterious outcomes and to develop potential treatments to battle pathophysiology-related diseases.

MicroRNAs (miR) are small RNA molecules that shape the cell transcriptome and proteome through regulation of mRNA stability and translation. Here, we examined their function as determinants of cell resistance to complement-dependent cytotoxicity (CDC). To achieve this goal, we compared the expression of microRNAs between complement-resistant and -sensitive K562 leukemia, Raji lymphoma, and HCT-116 colorectal carcinoma cells. Global microRNA array analysis identified miR-150, miR-328, and miR-616 as regulators of CDC resistance. Inhibition of miR-150 reduced resistance, whereas inhibition of miR-328 or miR-616 enhanced cell resistance. Treatment of K562 cells with a sublytic dose of complement was shown to rapidly increase miR-150, miR-328, and miR-616 expression. Protein targets of these microRNAs were analyzed in K562 cells by mass spectrometry–based proteomics. Expression of the complement membrane regulatory proteins CD46 and CD59 was significantly enhanced after inhibition of miR-328 and miR-616. Enrichment of proteins of mitochondria, known target organelles in CDC, was observed after miR-150, miR-328, and miR-616 inhibition. In conclusion, miR-150, miR-328, and miR-616 regulate cell resistance to CDC by modifying the expression of the membrane complement regulators CD46 and CD59 and the response of the mitochondria to complement lytic attack. These microRNAs may be considered targets for intervention in complement-associated diseases and in anticancer, complement-based therapy.

Proteomics technology aims to map the protein landscapes of biological samples, and it can be applied to a variety of samples, including cells, tissues, and body fluids. Because the proteins are the main functional molecules in the cells, their levels reflect much more accurately the cellular phenotype and the regulatory processes within them than gene levels, mutations, and even mRNA levels. With the advancement in the technology, it is possible now to obtain comprehensive views of the biological systems and to study large patient cohorts in a streamlined manner. In this review we discuss the technological advancements in mass spectrometry–based proteomics, which allow analysis of breast cancer tissue samples, leading to the first large-scale breast cancer proteomics studies. Furthermore, we discuss the technological developments in blood-based biomarker discovery, which provide the basis for future development of assays for routine clinical use. Although these are only the first steps in implementation of proteomics into the clinic, extensive collaborative work between these worlds will undoubtedly lead to major discoveries and advances in clinical practice.

Introduction One of the major obstacles in breast cancer treatment is its high degree of heterogeneity and ambiguous classification. The molecular subtypes and histological subtypes are routinely defined in the clinic to determine treatment modalities; however often these distinct subtypes co-exist in single tumours and therefore lead to treatment resistance. Here, we aim to delineate the proteomic landscapes of breast cancer intratumoral diversity based on clinical-pathological parameters using mass spectrometry-based microproteomics. Material and methods Using histopathological analysis of immunostained formalin fixed paraffin embedded (FFPE) tissues, we assembled a cohort of more than 200 tumour regions comprising of different histopathological and molecular subtypes that originate from 22 patients. Cancer cell subpopulations were harvested using laser-capture microdissection (LCM) and labelled by TMT 10-plex. After protein digestion, peptides were analysed using high resolution quantitative mass spectrometry followed by computational analysis with MaxQuant. Results and discussions Topological proteomic analysis revealed the proteomic portraits that are associated with each cancer cell subpopulation, with the association to its receptor expression level and histological characteristics. We found a clear proteomic distinction between moderately and poorly differentiated regions: moderately differentiated regions were primarily segregated based on their histological subtypes such as micropapillary vs. invasive ductal carcinoma-no special type, while poorly differentiated tumour regions were distinct based on the differential expression of the oestrogen, progesterone and Her2 receptors. We identified the significantly changing proteins between these subtypes, including significant differences in metabolic enzyme profiles between co-existing subtypes. For example, we found upregulated pentose-phosphate pathway in progesterone receptor positive regions and upregulated proline/arginine biosynthesis pathways in tumour regions with micropapillary features. Conclusion These findings suggest that integrated topo-proteomic landscape of inter- and intratumoral heterogeneity can serve as a platform for deciphering the mechanisms underlying tumorigenesis, distinct metabolic profiles of cancer cell subpopulations, cancer evolution, and therapy resistance.

Summary Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.