The oestrogen paradox: a hypothesis

Epidemiological and observational studies suggest that oestrogens, when used as hormonal therapy in post-menopausal women, can increase the risk of breast cancer if used long term. However, more recent data suggest that short-term use in sub-groups of post-menopausal women significantly decreases the risk of breast cancer. This beneficial effect is also observed when high-dose oestrogen is administered to post-menopausal women with breast cancer to cause tumour regression, a phenomenon which commonly occurs. We consider these divergent responses to oestrogen to represent a "paradox". Data from our own and other investigative groups suggest a hypothesis to explain this paradox. Deprivation of oestradiol in model systems causes cells to adapt and to undergo apoptosis in response to oestrogen. This occurs through the Fas/Fas ligand death receptor pathway and through alterations in apoptotic mechanisms mediated by mitochondria. This process of programmed cell death may explain the regression of established breast cancer with oestrogen administration and the diminution in the rate of new breast cancer diagnoses recently reported. Our hypothesis is based upon pathological data indicating the presence of a "reservoir" of undiagnosed breast cancer in the population of women who would be starting on oestrogens as menopausal hormonal therapy. The long-term increased risk of breast cancer may then reflect different mechanisms. Oestrogens can cause mutations through enhancement of the rate of cell division and concomitantly the error rate in DNA replication. In addition, oestrogens can be metabolised to directly genotoxic compounds. These carcinogenic processes take much longer, since a number of mutations must accumulate before resulting in breast cancer. These hypotheses regarding oestrogen-induced apoptosis in the short term and carcinogenesis in the long term now require rigorous verification but would serve to explain the "oestrogen paradox".     

Dissecting tumor responsiveness to immunotherapy: the experience of peptide-based melanoma vaccines

Recent years have witnessed important breakthroughs in our understanding of tumor immunology. A variety of immunotherapeutic strategies has shown that immune manipulation can induce the regression of established cancer in humans. The identification of the genes encoding tumor-associated antigens (TAA) and the development of means for immunizing against these antigens have opened new avenues for the development of an effective anticancer immunotherapy. However, an efficient immune response against tumor requires an intricate cross-talk between cancer and immune system cells, which is still poorly understood. Only when the molecular basis underlying tumor susceptibility to an immune response is deciphered could new therapeutic strategies be designed to fit biologically defined mechanisms of cancer immune rejection. In this article, we address some of the critical issues that have been identified in cancer immunotherapy, in part from our own studies on immune therapies in melanoma patients treated with peptide-based vaccination regimens. This is not meant to be a comprehensive overview of the immunological phenomena accompanying cancer patient vaccination but rather emphasizes some emergent findings, puzzling controversies and unanswered questions that characterize this complex field of oncology. In addition to reviewing the main immunological concepts underlying peptide-based vaccination, we also review the available data regarding naturally occurring and therapeutically induced anticancer immune response, both at the peripheral and intratumoral level. The hypothesized role of innate immunity in predetermining tumor responsiveness to immunotherapeutic manipulation is also discussed.     

Impaired recall of CD8 memory T cells in immunologically privileged tissue

Foreign Ags that enter immunologically privileged sites such as the eye, brain, and testis persist for an extended period of time, whereas the same Ags are rapidly eliminated at conventional sites. Immune privilege, therefore, provides unwanted refuge for pathogens and tumor cells but is beneficial for the survival of allogeneic grafts. In this study, we asked whether memory T cells can eliminate foreign Ags deposited at an immunologically privileged site by studying CD8 memory T cell-mediated rejection of pancreatic islet allografts placed either in the testis (a privileged organ) or under the kidney capsule (a nonprivileged site) of diabetic mice. We found that CD8 memory T cells reject intratesticular grafts at a significantly slower rate than the rejection of intrarenal grafts. Delayed graft rejection in the testis was not due to reduced homing or proliferation of memory T cells but due to their increased apoptosis at that site. Apoptosis was mediated by the combined actions of two TNFR family members that are up-regulated on activated memory T cells, Fas, and CD30. Therefore, memory T cells survey immunologically privileged tissues but are subject to the immunosuppressive mechanisms present at these sites.     

Epithelial to mesenchymal transition during gastrulation: An embryological view

Gastrulation is a developmental process to generate the mesoderm and endoderm from the ectoderm, of which the epithelial to mesenchymal transition (EMT) is generally considered to be a critical component. Due to increasing evidence for the involvement of EMT in cancer biology, a renewed interest is seen in using in vivo models, such as gastrulation, for studying molecular mechanisms underlying EMT. The intersection of EMT and gastrulation research promises novel mechanistic insight, but also creates some confusion. Here we discuss, from an embryological perspective, the involvement of EMT in mesoderm formation during gastrulation in triploblastic animals. Both gastrulation and EMT exhibit remarkable variations in different organisms, and no conserved role for EMT during gastrulation is evident. We propose that a ‘broken-down’ model, in which these two processes are considered to be a collective sum of separately regulated steps, may provide a better framework for studying molecular mechanisms of the EMT process in gastrulation, and in other developmental and pathological settings.     

Sources of spontaneous mutagenesis in bacteria

Mutations in an organism’s genome can arise spontaneously, that is, in the absence of exogenous stress and prior to selection. Mutations are often neutral or deleterious to individual fitness but can also provide genetic diversity driving evolution. Mutagenesis in bacteria contributes to the already serious and growing problem of antibiotic resistance. However, the negative impacts of spontaneous mutagenesis on human health are not limited to bacterial antibiotic resistance. Spontaneous mutations also underlie tumorigenesis and evolution of drug resistance. To better understand the causes of genetic change and how they may be manipulated in order to curb antibiotic resistance or the development of cancer, we must acquire a mechanistic understanding of the major sources of mutagenesis. Bacterial systems are particularly well-suited to studying mutagenesis because of their fast growth rate and the panoply of available experimental tools, but efforts to understand mutagenic mechanisms can be complicated by the experimental system employed. Here, we review our current understanding of mutagenic mechanisms in bacteria and describe the methods used to study mutagenesis in bacterial systems.     

Single-cell Analysis Technologies for Immuno-oncology Research: from Mechanistic Delineation to Biomarker Discovery

The successes with immune checkpoint blockade(ICB) and chimeric antigen receptor(CAR)-T-cell therapy in treating multiple cancer types have established immunotherapy as a powerful curative option for patients with advanced cancers. Unfortunately, many patients do not derive benefit or long-term responses, highlighting a pressing need to perform complete investigation of the underlying mechanisms and the immunotherapy-induced tumor regression or rejection.In recent years, a large number of single-cell technologies have leveraged advances in characterizing immune system, profiling tumor microenvironment, and identifying cellular heterogeneity, which establish the foundations for lifting the veil on the comprehensive crosstalk between cancer and immune system during immunotherapies. In this review, we introduce the applications of the most widely used single-cell technologies in furthering our understanding of immunotherapies in terms of underlying mechanisms and their association with therapeutic outcomes. We also discuss how single-cell analyses help to deliver new insights into biomarker discovery to predict patient response rate, monitor acquired resistance, and support prophylactic strategy development for toxicity management. Finally, we provide an overview of applying cutting-edge single-cell spatial-omics to point out the heterogeneity of tumor–immune interactions at higher level that can ultimately guide to the rational design of next-generation immunotherapies.     

Whole genome plasticity in pathogenic bacteria

The exploitation of bacterial genome sequences has so far provided a wealth of new general information about the genetic diversity of bacteria, such as that of many pathogens. Comparative genomics uncovered many genome variations in closely related bacteria and revealed basic principles involved in bacterial diversification, improving our knowledge of the evolution of bacterial pathogens. A correlation between metabolic versatility and genome size has become evident. The degenerated life styles of obligate intracellular pathogens correlate with significantly reduced genome sizes, a phenomenon that has been termed "evolution by reduction". These mechanisms can permanently alter bacterial genotypes and result in adaptation to their environment by genome optimization. In this review, we summarize the recent results of genome-wide approaches to studying the genetic diversity of pathogenic bacteria that indicate that the acquisition of DNA and the loss of genetic information are two important mechanisms that contribute to strain-specific differences in genome content.     

BCR-ABL Gene Expression Is Required for Its Mutations in a Novel KCL-22 Cell Culture Model for Acquired Resistance of Chronic Myelogenous Leukemia

Acquired resistance through genetic mutations is a common phenomenon in several cancer therapies using molecularly targeted drugs, best exemplified by the BCR-ABL inhibitor imatinib in treating chronic myelogenous leukemia (CML). Overcoming acquired resistance is a daunting therapeutic challenge, and little is known about how these mutations evolve. To facilitate understanding the resistance mechanisms, we developed a novel culture model for CML acquired resistance in which the CML cell line KCL-22, following initial response to imatinib, develops resistant T315I BCR-ABL mutation. We demonstrate that the emergence of BCR-ABL mutations do not require pre-existing BCR-ABL mutations derived from the original patient as the subclones of KCL-22 cells can form various BCR-ABL mutations upon imatinib treatment. BCR-ABL mutation rates vary from cell clone to clone and passages, in contrast to the relatively stable mutation rate of the hypoxanthine-guanine phosphoribosyltransferase gene. Strikingly, development of BCR-ABL mutations depends on its gene expression because BCR-ABL knockdown completely blocks KCL-22 cell relapse on imatinib and acquisition of mutations. We further show that the endogenous BCR-ABL locus has significantly higher mutagenesis potential than the transduced randomly integrated BCR-ABL cDNA. Our study suggests important roles of BCR-ABL gene expression and its native chromosomal locus for acquisition of BCR-ABL mutations and provides a new tool for further studying resistance mechanisms.     

Anti-PD1 therapy induces lymphocyte-derived exosomal miRNA-4315 release inhibiting Bim-mediated apoptosis of tumor cells

Anti-PD1 immunotherapy, as a single agent or in combination with standard chemotherapies, has significantly improved the outcome of many patients with cancers. However, resistance to anti-PD1 antibodies often decreases the long-term therapeutic benefits. Despite this observation in clinical practice, the molecular mechanisms associated with resistance to anti-PD1 antibody therapy have not yet been elucidated. To identify the mechanisms of resistance associated with anti-PD1 antibody therapy, we developed cellular models including purified T cells and different cancer cell lines from glioblastoma, lung adenocarcinoma, breast cancer and ovarian carcinoma. A murine model of lung cancer was also used. Longitudinal blood samples of patients treated with anti-PD1 therapy were also used to perform a proof-of-concept study of our findings. We found that anti-PD1 exposure of T-cell promotes an enrichment of exosomal miRNA-4315. We also noted that exosomal miRNA-4315 induced a phenomenon of apopto-resistance to conventional chemotherapies in cancer cells receiving exosomal miRNA-4315. At molecular level, we discern that the apopto-resistance phenomenon was associated with the miRNA-4315-mediated downregulation of Bim, a proapoptotic protein. In cellular and mice models, we observed that the BH3 mimetic agent ABT263 circumvented this resistance. A longitudinal study using patient blood showed that miRNA-4315 and cytochrome c can be used to define the time period during which the addition of ABT263 therapy may effectively increase cancer cell death and bypass anti-PD1 resistance.This work provides a blood biomarker (exosomal miRNA-4315) for patient stratification developing a phenomenon of resistance to anti-PD1 antibody therapy and also identifies a therapeutic alternative (the use of a BH3 mimetic drug) to limit this resistance phenomenon.Subject terms: Cancer, miRNAs     

Phellinus Linteus Extract Sensitizes Advanced Prostate Cancer Cells to Apoptosis in Athymic Nude Mice

Phellinus linteus (PL) mushroom possesses anti-tumor property. We previously reported that the treatment with PL caused cultured human prostate cancer cells to undergo apoptosis. To further studying the mechanisms of PL-mediated apoptosis, we performed xenograft assay, together with in vitro assays, to evaluate the effect of PL on the genesis and progression of the tumors formed from the inoculation of prostate cancer PC3 or DU145 cells. After the inoculation, nude mice were injected with PL every two days for 12 days. Although PL treatment did not prevent the formation of the inoculated tumors, the growth rate of the tumors after PL treatment was dramatically attenuated. We then tested the effect of PL on the tumors 12 days after the inoculation. After inoculated tumors reached a certain size, PL was administrated to the mice by subcutaneous injection. The histochemistry or immunochemistry analysis showed that apoptosis occurred with the activation of caspase 3 in the tumors formed by inoculating prostate cancer DU145 or PC3 cells. The data was in a good agreement with that from cultured cells. Thus, our in vivo study suggests that PL not only is able to attenuate tumor growth, but also to cause tumor regression by inducing apoptosis.     

Adeno-Associated Virus Vector Mediated Expression of an Oncogenic Retroviral Envelope Protein Induces Lung Adenocarcinomas in Immunocompetent Mice

Lung cancer is the most common cause of cancer-related death worldwide. A poor overall survival rate of 16% necessitates the need for novel treatment strategies. Mouse models of lung cancer are important tools for analyzing the significance of somatic mutations in the initiation and progression of lung cancer. Of additional importance, however, are animal models of virally induced cancers. JSRV is a simple betaretrovirus that causes contagious lung cancer in sheep known as ovine pulmonary adenocarcinoma and closely resembles human lung adenocarcinoma. Previously we showed that expression of the JSRV envelope (Env) from an AAV vector induced lung tumors in immunodeficient mice, but not in immunocompetent mice. Because of the importance of studying lung cancer in the context of an intact immune system we sought to improve our mouse model. In this report, we employed the use of a strong JSRV enhancer-promoter combination to express Env at high levels and demonstrate for the first time, lung tumor induction in immunocompetent mice. This occurred despite a robust Env-specific antibody-mediated immune response. The PI3K/Akt and MAPK pathways were activated in both immunocompetent and immunodeficient mice, however, differential activation of PTEN, GSKα, p70S6K, p38MAPK, ATF2 and STAT5 was observed. A JSRV Env lung tumor-derived cell line was shown to have a similar signal transduction activation profile as Env-induced lung tumors in C57BL/6 mice. Given the similarities between our model and pulmonary adenocarcinomas in humans, and the ease with which tumors can be induced in any transgenic mouse, this system can be used to uncover novel mechanisms involved lung tumorigenesis.     

Mechanisms of the antitumor responses and host-versus-graft reactions induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: a critical role for recipient CD4+ T cells and recipient leukocyte infusion-derived IFN-gamma-producing CD8+ T cells

Surprisingly, antitumor responses can occur in patients who reject donor grafts following nonmyeloablative hemopoietic cell transplantation. In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism, IFN-gamma production, and antitumor responses against host-type tumors. However, the mechanisms behind this phenomenon remain to be determined. We now demonstrate that the effects of RLI are mediated by distinct and complex mechanisms. Donor marrow rejection is induced by RLI-derived alloactivated T cells, which activate non-RLI-derived, recipient IFN-gamma-producing cells. RLI-derived CD8 T cells induce the production of IFN-gamma by both RLI and non-RLI-derived recipient cells. The antitumor responses of RLI involve mainly RLI-derived IFN-gamma-producing CD8 T cells and recipient-derived CD4 T cells and do not involve donor T cells. The pathways of donor marrow and tumor rejection lead to the development of tumor-specific cell-mediated cytotoxic responses that are not due to bystander killing by alloreactive T cells.     

Transient exposure to androgens induces a remarkable self-sustained quiescent state in dispersed prostate cancer cells

Cellular quiescence is a reversible cell growth arrest that is often assumed to require a persistence of non-permissive external growth conditions for its maintenance. In this work, we showed that androgen could induce a quiescent state that is self-sustained in a cell-autonomous manner through a “hit and run” mechanism in androgen receptor-expressing prostate cancer cells. This phenomenon required the set-up of a sustained redox imbalance and TGFβ/BMP signaling that were dependent on culturing cells at low density. At medium cell density, androgens failed to induce such a self-sustained quiescent state, which correlated with a lesser induction of cell redox imbalance and oxidative stress markers like CDKN1A. These effects of androgens could be mimicked by transient overexpression of CDKN1A that triggered its own expression and a sustained SMAD phosphorylation in cells cultured at low cell density. Overall, our data suggest that self-sustained but fully reversible quiescent states might constitute a general response of dispersed cancer cells to stress conditions.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

Telomerase-mediated lifespan extension of human bronchial cells does not affect hexavalent chromium-induced cytotoxicity or genotoxicity

Hexavalent chromium (Cr(VI)) is a metal of increasing public health concern, as exposure to it is widespread and it is a well-established cause of human bronchial carcinomas and fibrosarcomas. The water-insoluble Cr(VI) salts are potent carcinogens compared to the water soluble salts; yet the genotoxic mechanisms of both may be mediated by soluble Cr(VI) ions. Currently, these mechanisms are poorly understood. Emerging evidence suggests that initial cell culture models used to study the general toxicity of Cr(VI) may be suboptimal for investigating mechanisms specific to human bronchial cells. Accordingly, we have developed a new model system of human bronchial cells by introducing hTERT, the catalytic subunit of human telomerase, into primary human bronchial fibroblasts (PHBF). We have isolated a stable, clonally derived cell line, WHTBF-6, that demonstrate reconstitution of telomerase activity and maintenance of telomere lengths with increasing culture age. WHTBF-6 has been characterized as having an extended in vitro lifespan, a normal growth rate, a normal diploid karyotype that is maintained over time, and exhibits serum-dependent contact-inhibited anchorage-dependent growth. Moreover, we find that both particulate and soluble hexavalent chromium induce a pattern and degree of cytotoxicity and clastogenicity in WHTBF-6 that is similar to the parental PHBF cells. Because telomerase does not compromise growth or the response to Cr(VI), our results indicate that this is an excellent system for studying the mechanisms of Cr(VI) and potentially other carcinogens implicated in the development of lung cancer.     

Modeling adaptive drug resistance of colorectal cancer and therapeutic interventions with tumor spheroids

Drug resistance is a major barrier against successful treatments of cancer patients. Various intrinsic mechanisms and adaptive responses of tumor cells to cancer drugs often lead to failure of treatments and tumor relapse. Understanding mechanisms of cancer drug resistance is critical to develop effective treatments with sustained anti-tumor effects. Three-dimensional cultures of cancer cells known as spheroids present a biologically relevant model of avascular tumors and have been increasingly incorporated in tumor biology and cancer drug discovery studies. In this review, we discuss several recent studies from our group that utilized colorectal tumor spheroids to investigate responses of cancer cells to cytotoxic and molecularly targeted drugs and uncover mechanisms of drug resistance. We highlight our findings from both short-term, one-time treatments and long-term, cyclic treatments of tumor spheroids and discuss mechanisms of adaptation of cancer cells to the treatments. Guided by mechanisms of resistance, we demonstrate the feasibility of designing specific drug combinations to effectively block growth and resistance of cancer cells in spheroid cultures. Finally, we conclude with our perspectives on the utility of three-dimensional tumor models and their shortcomings and advantages for phenotypic and mechanistic studies of cancer drug resistance.     

The Ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of IAPs

A better understanding of the mechanisms through which anticancer drugs exert their effects is essential to improve combination therapies. While studying how genotoxic stress kills cancer cells, we discovered a large ～2MDa cell death-inducing platform, referred to as "Ripoptosome." It contains the core components RIP1, FADD, and caspase-8, and assembles in response to genotoxic stress-induced depletion of XIAP, cIAP1 and cIAP2. Importantly, it forms independently of TNF, CD95L/FASL, TRAIL, death-receptors, and mitochondrial pathways. It also forms upon Smac-mimetic (SM) treatment without involvement of autocrine TNF. Ripoptosome assembly requires RIP1's kinase activity and can stimulate caspase-8-mediated apoptosis as well as caspase-independent necrosis. It is negatively regulated by FLIP, cIAP1, cIAP2, and XIAP. Mechanistically, IAPs target components of this complex for ubiquitylation and inactivation. Moreover, we find that etoposide-stimulated Ripoptosome formation converts proinflammatory cytokines into prodeath signals. Together, our observations shed new light on fundamental mechanisms by which chemotherapeutics may kill cancer cells.     

Copying You Copying Me: Interpersonal Motor Co-Ordination Influences Automatic Imitation

Moving in a co-ordinated fashion with another individual changes our behaviour towards them; we tend to like them more, find them more attractive, and are more willing to co-operate with them. It is generally assumed that this effect on behaviour results from alterations in representations of self and others. Specifically, through neurophysiological perception-action matching mechanisms, interpersonal motor co-ordination (IMC) is believed to forge a neural coupling between actor and observer, which serves to blur boundaries in conceptual self-other representations and causes positive views of the self to be projected onto others. An investigation into this potential neural mechanism is lacking, however. Moreover, the specific components of IMC that might influence this mechanism have not yet been specified. In the present study we exploited a robust behavioural phenomenon – automatic imitation – to assess the degree to which IMC influences neural action observation-execution matching mechanisms. This revealed that automatic imitation is reduced when the actions of another individual are perceived to be synchronised in time, but are spatially incongruent, with our own. We interpret our findings as evidence that IMC does indeed exert an effect on neural perception-action matching mechanisms, but this serves to promote better self-other distinction. Our findings demonstrate that further investigation is required to understand the complex relationship between neural perception-action coupling, conceptual self-other representations, and social behaviour.