Blocking PGE2-induced tumour repopulation abrogates bladder cancer chemoresistance.

Cytotoxic chemotherapy is effective in debulking tumour masses initially; however, in some patients tumours become progressively unresponsive after multiple treatment cycles. Previous studies have demonstrated that cancer stem cells (CSCs) are selectively enriched after chemotherapy through enhanced survival. Here we reveal a new mechanism by which bladder CSCs actively contribute to therapeutic resistance via an unexpected proliferative response to repopulate residual tumours between chemotherapy cycles, using human bladder cancer xenografts. Further analyses demonstrate the recruitment of a quiescent label-retaining pool of CSCs into cell division in response to chemotherapy-induced damages, similar to mobilization of normal stem cells during wound repair. While chemotherapy effectively induces apoptosis, associated prostaglandin E2 (PGE2) release paradoxically promotes neighbouring CSC repopulation. This repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. In vivo administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation.

Three differentiation states risk-stratify bladder cancer into distinct subtypes.

Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.

Reactive oxygen species and hematopoietic stem cell senescence.

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism through self-renewal, proliferation, differentiation, and mobilization. Their functions can be affected by reactive oxygen species (ROS) that are produced endogenously through cellular metabolism or after exposure to exogenous stress. At physiological levels, ROS function as signal molecules which can regulate a variety of cellular functions, including HSC proliferation, differentiation, and mobilization. However, an abnormal increase in ROS production occurs under various pathological conditions, which can inhibit HSC self-renewal and induce HSC senescence, resulting in premature exhaustion of HSCs and hematopoietic dysfunction. This review aims to provide a summary of a number of recent findings regarding the cellular sources of ROS in HSCs and the mechanisms of action whereby ROS induce HSC senescence. In particular, we highlight the roles of the p38 mitogen-activated protein kinase (p38)-p16(Ink4a) (p16) pathway in mediating ROS-induced HSC senescence.

Mn(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin mitigates total body irradiation-induced long-term bone marrow suppression.

Our recent studies showed that total body irradiation (TBI) induces long-term bone marrow (BM) suppression in part by induction of hematopoietic stem cell (HSC) senescence through reactive oxygen species (ROS). In this study, we examined if Mn(III) meso-tetrakis-(N-ethylpyridinium-2-yl) porphyrin (MnTE), a superoxide dismutase mimetic and potent antioxidant, can mitigate TBI-induced long-term BM injury in a mouse model. Our results showed that post-TBI treatment with MnTE significantly inhibited the increases in ROS production and DNA damage in HSCs and the reduction in HSC frequency and clonogenic function induced by TBI. In fact, the clonogenic function of HSCs from irradiated mice after MnTE treatment was comparable to that of HSCs from normal controls on a per-HSC basis, suggesting that MnTE treatment inhibited the induction of HSC senescence by TBI. This suggestion is supported by the finding that MnTE treatment also reduced the expression of p16(Ink4a) (p16) mRNA in HSCs induced by TBI and improved the long-term and multilineage engraftment of irradiated HSCs after transplantation. Therefore, the results from this study demonstrate that MnTE has the potential to be used as a therapeutic agent to mitigate TBI-induced long-term BM suppression by inhibiting ionizing radiation-induced HSC senescence through the ROS-p16 pathway.

IKKß regulates the repair of DNA double-strand breaks induced by ionizing radiation in MCF-7 breast cancer cells.

Activation of the IKK-NFκB pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NFκB. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NFκB pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NFκB pathway with a specific IKKß inhibitor significantly reduced the repair of IR-induced DSBs in MCF-7 cells. The repair of DSBs was also significantly inhibited by silencing IKKß expression with IKKß shRNA. However, down-regulation of IKKα expression with IKKα shRNA had no significant effect on the repair of IR-induced DSBs. Similar findings were also observed in IKKα and/or IKKß knockout mouse embryonic fibroblasts (MEFs). More importantly, inhibition of IKKß with an inhibitor or down-regulation of IKKß with IKKß shRNA sensitized MCF-7 cells to IR-induced clonogenic cell death. DSB repair function and resistance to IR were completely restored by IKKß reconstitution in IKKß-knockdown MCF-7 cells. These findings demonstrate that IKKß can regulate the repair of DSBs, a previously undescribed and important IKKß kinase function; and inhibition of DSB repair may contribute to cance cell radiosensitization induced by IKKß inhibition. As such, specific inhibition of IKKß may represents a more effective approach to sensitize cancer cells to radiotherapy.

NADPH oxidase inhibition attenuates total body irradiation-induced haematopoietic genomic instability.

Ionising radiation (IR) is a known carcinogen and poses a significant risk to the haematopoietic system for the development of leukaemia in part by induction of genomic instability. Induction of chronic oxidative stress has been assumed to play an important role in mediating the effect of IR on the haematopoietic system. However, there was no direct evidence to support this hypothesis prior to our studies. In our recent studies, we showed that exposure of mice to total body irradiation (TBI) induces persistent oxidative stress selectively in haematopoietic stem cells (HSCs) at least in part via up-regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4. Now, we found that post-TBI treatment with diphenylene iodonium (DPI), a pan NOX inhibitor, not only significantly reduces TBI-induced increases in reactive oxygen species (ROS) production, oxidative DNA damage and DNA double-strand breaks in HSCs but also dramatically decreases the number of cells with unstable chromosomal aberrations in the clonal progeny of irradiated HSCs. The effects of DPI are comparable to Mn (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin, a superoxide dismutase mimetic and a potent antioxidant. These findings demonstrate that increased production of ROS by NOX in HSCs mediates the induction of haematopoietic genomic instability by IR and that NOX may represent a novel molecular target to inhibit TBI-induced genomic instability.

Total body irradiation causes residual bone marrow injury by induction of persistent oxidative stress in murine hematopoietic stem cells.

Ionizing radiation (IR) and/or chemotherapy causes not only acute tissue damage but also late effects including long-term (or residual) bone marrow (BM) injury. The induction of residual BM injury is primarily attributable to the induction of hematopoietic stem cell (HSC) senescence. However, the molecular mechanisms by which IR and/or chemotherapy induces HSC senescence have not been clearly defined, nor has an effective treatment been developed to ameliorate the injury. Thus, we investigated these mechanisms in this study. The results from this study show that exposure of mice to a sublethal dose of total body irradiation (TBI) induced a persistent increase in reactive oxygen species (ROS) production in HSCs only. The induction of chronic oxidative stress in HSCs was associated with sustained increases in oxidative DNA damage, DNA double-strand breaks (DSBs), inhibition of HSC clonogenic function, and induction of HSC senescence but not apoptosis. Treatment of the irradiated mice with N-acetylcysteine after TBI significantly attenuated IR-induced inhibition of HSC clonogenic function and reduction of HSC long-term engraftment after transplantation. The induction of chronic oxidative stress in HSCs by TBI is probably attributable to the up-regulation of NADPH oxidase 4 (NOX4), because irradiated HSCs expressed an increased level of NOX4, and inhibition of NOX activity with diphenylene iodonium but not apocynin significantly reduced TBI-induced increases in ROS production, oxidative DNA damage, and DNA DSBs in HSCs and dramatically improved HSC clonogenic function. These findings provide the foremost direct evidence demonstrating that TBI selectively induces chronic oxidative stress in HSCs at least in part via up-regulation of NOX4, which leads to the induction of HSC senescence and residual BM injury.

Stem cells, DNA damage, ageing and cancer.

A comprehensive knowledge of adult stem cells not only sheds light on their significant roles in many fundamental biological process, but also on their implications in a variety of deleterious disorders including leukemia, lymphoma, ageing and other relevant degenerative disorders. Elucidating the diverse signaling pathways in stem cells paves the way for development of targeted therapeutic approaches against degenerative disorders. Despite emerging studies, our understanding of the key molecular machineries that profoundly influence key stem cell properties is poor. This review discusses the seminal aspects of stem cell self-renewal mechanisms with an emphasis on various influencing factors, including DNA damage, and intrinsic and extrinsic factors that govern the significant characteristics of tissue stem cells under normal and stress conditions. Also outlined are intriguing aspects of how molecular interventions in key stem cell pathways could be exploited to develop novel therapies against stem cell disorders.