SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGβ/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGβ/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFβ/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.     

Model of Tumor Dormancy/Recurrence after Short-Term Chemotherapy

Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. We have developed an in vitro model of tumor dormancy/recurrence. Short-term exposure of tumor cells (breast or prostate) to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from “recurrent” colonies exhibit increased chemotherapy resistance, similar to the therapy resistance of recurrent tumors in cancer patients. Previous studies using long-term chemotherapy selection models identified acquired mutations that drive tumor resistance. In contrast, our short term chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that can resume growth after drug removal. Studying unique signaling pathways in dormant tumor cells enriched by short-term chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.     

细胞休眠在肿瘤耐药和复发中的作用（英文）

Despite progresses achieved in the therapy of tumors, the prognosis of patients is still limited by reccurence of residual tumor cells. Cancer cell dormancy plays a pivotal role in cancer relapse and drug resistance. In recent years, tumor cells undergoing EMT(epithelial-mesenchymal transition), CSCs(cancer stem cells) and CTCs(circulating tumor cells) are proved to share some common characteristics and show a cell cycle arrest phenotype. Thus, understanding the dormant stage of tumor cells could facilitate us in discovering ways to accelerate the development of tumor therapy and prevent its reccurence. In this review, we summarize the specific process of tumor cell dormancy induced by pharmacotherapy, and consider that dormancy is an initiative response rather than a passive defense to cytotoxicity. Besides, we probe into the mechanisms of tumor cell dormancy-mediated drug resistance, anticipating paving a way to target dormant tumor cells and result in better clinical outcomes.     

细胞休眠在肿瘤耐药和复发中的作用

目前对于肿瘤的药物治疗已经取得了一定的进展,然而,治疗后残存肿瘤细胞的复发仍然是导致肿瘤治疗失败的主要原因．肿瘤细胞休眠在肿瘤复发及耐药中发挥着重要的作用．近年来,研究发现上皮间质转化(epithelial-mesenchymal transition,EMT)的肿瘤细胞,肿瘤干细胞(cancer stem cells,CSCs)以及循环肿瘤细胞(circulating tumor cells,CTCs)都显示出细胞周期阻滞的状态．因此,对于休眠阶段肿瘤细胞的研究将可能促进肿瘤的治疗及预防肿瘤的复发．本文总结了药物治疗诱导肿瘤细胞发生休眠的具体过程,同时认为休眠是肿瘤细胞面对药物治疗所采取的主动防御措施,而非被动逃避过程．探究药物诱导性肿瘤细胞的休眠机制对于靶向休眠肿瘤细胞治疗及提高临床治疗效果都具有非常重要的意义．     

Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer

Breast cancers can recur after removal of the primary tumor and treatment to eliminate remaining tumor cells. Recurrence may occur after long periods of time during which there are no clinical symptoms. Tumor cell dormancy may explain these prolonged periods of asymptomatic residual disease and treatment resistance. We generated a dormancy gene signature from published experimental models and applied it to both breast cancer cell line expression data as well as four published clinical studies of primary breast cancers. We found that estrogen receptor (ER) positive breast cell lines and primary tumors have significantly higher dormancy signature scores (P<0.0000001) than ER- cell lines and tumors. In addition, a stratified analysis combining all ER+ tumors in four studies indicated 2.1 times higher hazard of recurrence among patients whose tumors had low dormancy scores (LDS) compared to those whose tumors had high dormancy scores (HDS) (p<0.000005). The trend was shown in all four individual studies. Suppression of two dormancy genes, BHLHE41 and NR2F1, resulted in increased in vivo growth of ER positive MCF7 cells. The patient data analysis suggests that disseminated ER positive tumor cells carrying a dormancy signature are more likely to undergo prolonged dormancy before resuming metastatic growth. Furthermore, genes identified with this approach might provide insight into the mechanisms of dormancy onset and maintenance as well as dormancy models using human breast cancer cell lines.     

RelE-mediated dormancy is enhanced at high cell density in Escherichia coli

Bacteria show remarkable adaptability under several stressful conditions by shifting themselves into a dormant state. Less is known, however, about the mechanism underlying the cell transition to dormancy. Here, we report that the transition to dormant states is mediated by one of the major toxin-antitoxin systems, RelEB, in a cell density-dependent manner in Escherichia coli K-12 MG1655. We constructed a strain, IKA121, which expresses the toxin RelE in the presence of rhamnose and lacks chromosomal relBE and rhaBAD. With this strain, we demonstrated that RelE-mediated dormancy is enhanced at high cell densities compared to that at low cell densities. The initiation of expression of the antitoxin RelB from a plasmid, pCA24N, reversed RelE-mediated dormancy in bacterial cultures. The activation of RelE increased the appearance of persister cells against β-lactams, quinolones, and aminoglycosides, and more persister cells appeared at high cell densities than at low cell densities. Further analysis indicated that amino acid starvation and an uncharacterized extracellular heat-labile substance promote RelE-mediated dormancy. This is a first report on the induction of RelE-mediated dormancy by high cell density. This work establishes a population-based dormancy mechanism to help explain E. coli survival in stressful environments.     

Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast

Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl(2) at 5muM concentration, corresponding to the IC(50) for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.     

Dormancy of Solitary Metastatic Cells

After arriving in a secondary site metastatic cells either begin proliferating, undergo apoptosis or remain as solitary dormant cells. The process of metastasis, although dangerous, is extremely inefficient with the majority of the cells undergoing apoptosis and thus becoming clinically irrelevant. Of the cells that begin proliferating, the few that make it past the micrometastasis stage may be of immediate clinical relevance. Dormant cells, while not of immediate clinical concern, are believed to be at least in part responsible for cancer recurrence that can occur decades after apparently successful initial treatment. Dormant solitary cells are different from “dormant” micrometastases, in which active proliferation is balanced by apoptosis. The mechanisms of cell cycle regulation and the function of the molecules regulating this process are well understood. However, there is relatively little known about the mechanisms controlling cell cycle regulation and dormancy of solitary metastatic cells. There are several inherent difficulties impeding the study of solitary cells. This review paper will examine the models used in the study of dormant solitary metastatic cells, methods of imaging and studying these cells, the molecular mechanisms believed to be responsible for solitary cell dormancy, and finally the unique treatment challenges posed by these cells.     

Autophagy,senescence and tumor dormancy in cancer therapy

The relationships between autophagy and apoptosis have been examined quite extensively and have often been shown to be reciprocally regulated responses to stresses such as exposure of the tumor cells to chemotherapeutic drugs and radiation. However, there is now evidence that autophagy may also play a role in tumor dormancy. Given that tumor dormancy and disease recurrence are poorly understood phenomenon which are nevertheless critical elements of patient morbidity and mortality, this commentary develops the postulate that autophagy and senescence may be related responses that influence the capacity of the tumor cell to maintain a prolonged state of growth arrest that can be succeeded by tumor regrowth and disease recurrence.     

Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.     

Glypican-3 (GPC3) inhibits metastasis development promoting dormancy in breast cancer cells by p38 MAPK pathway activation

GPC3 is a proteoglycan involved in the control of proliferation and survival, which has been linked to several tumor types. In this respect, we previously demonstrated that normal breast tissues exhibit high levels of GPC3, while its expression is diminished in tumors. However, the role of the GPC3 downregulation in breast cancer progression and its molecular and cellular operational machineries are not fully understood.In this study we showed that GPC3 reverts the epithelial-to-mesenchymal transition (EMT) underwent by mammary tumor cells, blocks metastatic spread and induces dormancy at secondary site. Using genetically modified murine breast cancer cell sublines, we demonstrated that the phospho-Erk/phospho-p38 ratio is lower in GPC3 reexpressing cells, while p21, p27 and SOX2 levels are higher, suggesting a dormant phenotype. In vivo metastasis assays confirmed that GPC3 reexpressing cells reduce their metastatic ability. Interestingly, the presence of dormant cells was evidenced in the lungs of inoculated mice. Dormant cells could reactivate their proliferative capacity, remain viable as well as tumorigenic, but they reentered in dormancy upon reaching secondary site. We also proved that GPC3 inhibits metastasis through p38 pathway activation. The in vivo inhibition of p38 induced an increase in cell invasion of GPC3 reexpressing orthotropic tumors as well as in spontaneous and experimental metastatic dissemination.In conclusion, our study shows that GPC3 returns mesenchymal-like breast cancer cells to an epithelial phenotype, impairs in vivo metastasis and induces tumor dormancy through p38 MAPK signaling activation. These results help to identify genetic determinants of dormancy and suggest the translational potential of research focusing in GPC3.     

Some implications of Scale Relativity theory in avascular stages of growth of solid tumors in the presence of an immune system response

We present a traveling-wave analysis of a reduced mathematical model describing the growth of a solid tumor in the presence of an immune system response in the framework of Scale Relativity theory. Attention is focused upon the attack of tumor cells by tumor-infiltrating cytotoxic lymphocytes (TICLs), in a small multicellular tumor, without necrosis and at some stage prior to (tumor-induced) angiogenesis. For a particular choice of parameters, the underlying system of partial differential equations is able to simulate the well-documented phenomenon of cancer dormancy and propagation of a perturbation in the tumor cell concentration by cnoidal modes, by depicting spatially heterogeneous tumor cell distributions that are characterized by a relatively small total number of tumor cells. This behavior is consistent with several immunomorphological investigations. Moreover, the alteration of certain parameters of the model is enough to induce soliton like modes and soliton packets into the system, which in turn result in tumor invasion in the form of a standard traveling wave. In the same framework of Scale Relativity theory, a very important feature of malignant tumors also results, that even in avascular stages they might propagate and invade healthy tissues, by means of a diffusion on a Newtonian fluid.     

Modeling progression in radiation-induced lung adenocarcinomas

Quantitative multistage carcinogenesis models are used in radiobiology to estimate cancer risks and latency periods (time from exposure to clinical cancer). Steps such as initiation, promotion and transformation have been modeled in detail. However, progression, a later step during which malignant cells can develop into clinical symptomatic cancer, has often been approximated simply as a fixed lag time. This approach discounts important stochastic mechanisms in progression and evidence on the high prevalence of dormant tumors. Modeling progression more accurately is therefore important for risk assessment. Unlike models of earlier steps, progression models can readily utilize not only experimental and epidemiological data but also clinical data such as the results of modern screening and imaging. Here, a stochastic progression model is presented. We describe, with minimal parameterization: the initial growth or extinction of a malignant clone after formation of a malignant cell; the likely dormancy caused, for example, by nutrient and oxygen deprivation; and possible escape from dormancy resulting in a clinical cancer. It is shown, using cohort simulations with parameters appropriate for lung adenocarcinomas, that incorporating such processes can dramatically lengthen predicted latency periods. Such long latency periods together with data on timing of radiation-induced cancers suggest that radiation may influence progression itself.     

Cancer dormancy. VII. A regulatory role for CD8+ T cells and IFN-gamma in establishing and maintaining the tumor-dormant state

Dormant tumor cells resistant to ablative cancer therapy represent a significant clinical obstacle due to later relapse. Experimentally, the murine B cell lymphoma (BCL1) is used as a model of tumor dormancy in mice vaccinated with the BCL1 Ig. Here, we used this model to explore the cellular mechanisms underlying dormancy. Our previous studies have demonstrated that T cell-mediated immunity is an important component in the regulation of tumor dormancy because Id-immune T cells adoptively transferred into passively immunized SCID mice challenged with BCL1 cells significantly increased the incidence and duration of the dormant state. We have extended these observations and demonstrate that CD8+, but not CD4+, T cells are required for the maintenance of dormancy in BCL1 Ig-immunized BALB/c mice. In parallel studies, the transfer of Id-immune CD8+ cells, but not Id-immune CD4+ cells, conferred significant protection to SCID mice passively immunized with nonprotective levels of polyclonal anti-Id and then challenged with BCL1 cells. Furthermore, the ability of CD8+ T cells to induce a state of dormancy in passively immunized SCID mice was completely abrogated by treatment with neutralizing alpha-IFN-gamma mAbs in vivo. In vitro studies demonstrated that IFN-gamma alone or in combination with reagents to cross-link the surface Ig induced both cell cycle arrest and apoptosis in a BCL1 cell line. Collectively, these data demonstrate a role for CD8+ T cells via endogenous production of IFN-gamma in collaboration with humoral immunity to both induce and maintain a state of tumor dormancy.     

Osteoblastic protein kinase D1 contributes to the prostate cancer cells dormancy via GAS6-circadian clock signaling

Disseminated prostate cancer (PCa) is known to have a strong propensity for bone marrow. These disseminated tumor cells (DTCs) can survive in bone marrow for years without obvious proliferation, while maintaining the ability to develop into metastatic lesions. However, how DTCs kept dormant and recur is still uncertain. Here, we focus on the role of osteoblastic protein kinase D1 (PKD1) in PCa (PC-3 and DU145) dormancy using co-culture experiments. Using flow cytometry, western blotting, and immunofluorescence, we observed that in co-cultures osteoblasts could induce a dormant state in PCa cells, which is manifested by a fewer cell divisions, a decrease Ki-67-positive populations and a lower ERK/p38 ratio. In contrast, silencing of PKD1 gene in osteoblasts impedes co-cultured prostate cancer cell's dormancy ability. Mechanismly, protein kinase D1 (PKD1) in osteoblasts induces PCa dormancy via activating CREB1, which promoting the expression and secretion of growth arrest specific 6 (GAS6). Furthermore, GAS6-induced dormancy signaling significantly increased the expression of core circadian clock molecules in PCa cells, and a negative correlation of circadian clock proteins (BMAL1, CLOCK and DEC2) with recurrence-free survival is observed in metastatic prostate cancer patients. Interestingly, the expression of cell cycle factors (p21, p27, CDK1 and PCNA) which regulated by circadian clock also upregulated in response to GAS6 stimulation. Taken together, we provide evidence that osteoblastic PKD1/CREB1/GAS6 signaling regulates cellular dormancy of PCa cells, and highlights the importance of circadian clock in PCa cells dormancy.     

Alterations in ultrastructure and subcellular localization of Ca2+ in poplar apical bud cells during the induction of dormancy

In poplar (Populus deltoides Bartr. ex Marsh), bud dormancyand freezing tolerance were concomitantly induced by short-day(SD) photoperiods. Ultrastructural changes and the alterationin subcellular localization of calcium in apical bud cells associatedwith dormancy development were investigated. During the developmentof dormancy, the thickness of cell walls increased significantly,the number of starch granules increased, and there was a significantaccumulation of storage proteins in the vacuoles of the apicalbud cells. The most striking change was the constriction andblockage of the plasmodesmata. It was demonstrated that antimonate precipitation is a reliabletechnique for studying subcellular localization of calcium inpoplar apical bud cells. Under the long day (LD) photoperiod,electron-dense calcium antimonate precipitates were mainly localizedin vacuoles, intercellular spaces and plastids. Some antimonateprecipitates were also found in the cell walls and at the entranceof the plasmodesmata. However, there were few Ca²⁺ depositsfound in the cytosol and nucleus. After 20 d of SD exposure,when development of bud dormancy was initiated, calcium depositsin intercellular spaces were decreased, whereas some depositswere found in the cytosol and nuclei. From 28–49 d ofSD exposure, while dormancy was developing, a large number ofCa²⁺ precipitates were found in the cytosol and nuclei. Whendeep dormancy was reached after 77 d of SD exposure, Ca²⁺ depositsbecame fewer in both cytosol and nuclei, whereas numerous depositswere again observed in the cell walls and in the intercellularspaces. These results suggest that under the influence of SDphotoperiods, there are alterations in subcellular Ca²⁺ localization,and changes in ultrastructure of apical bud cells during thedevelopment of dormancy. The constriction and blockage of plasmodesmatamay cause the cessation of symplastic transport, limit cellularcommunication and signal transduction between adjacent cells,which in turn may lead to events associated with growth cessationand dormancy development in buds. Key words: Poplar, apical bud cells, Ca²⁺ subcellular localization, dormancy     

Engineered <Emphasis Type="Italic">In Vitro</Emphasis> Models of Tumor Dormancy and Reactivation

Metastatic recurrence is a major hurdle to overcome for successful control of cancer-associated death. Residual tumor cells in the primary site, or disseminated tumor cells in secondary sites, can lie in a dormant state for long time periods, years to decades, before being reactivated into a proliferative growth state. The microenvironmental signals and biological mechanisms that mediate the fate of disseminated cancer cells with respect to cell death, single cell dormancy, tumor mass dormancy and metastatic growth, as well as the factors that induce reactivation, are discussed in this review. Emphasis is placed on engineered, in vitro, biomaterial-based approaches to model tumor dormancy and subsequent reactivation, with a focus on the roles of extracellular matrix, secondary cell types, biochemical signaling and drug treatment. A brief perspective of molecular targets and treatment approaches for dormant tumors is also presented. Advances in tissue-engineered platforms to induce, model, and monitor tumor dormancy and reactivation may provide much needed insight into the regulation of these processes and serve as drug discovery and testing platforms.     

Development and Characterization of a Preclinical Model of Breast Cancer Lung Micrometastatic to Macrometastatic Progression

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500–1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30–60 micrometastases in the lung (with many animals also having 2–30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.