Breast Cancer Stem Cells, Pathways and Therapeutic Perspectives 2011

The evidence for the existence of a heterogeneous population of cancer stem cells (CSCs) responsible for the initiation and maintenance of cancer has been characterized for several tumors recently. Purification and molecular characterization of normal human mammary stem cells from cultured mammospheres has been achieved, providing evidence supporting a model in which breast tumor heterogeneity is a reflection of a number of CSC-like cells in the tumor. A number of experimental methodologies have been developed to characterize epithelial stem cells, including the expression of cell surface or intracellular markers, mammosphere formation, exclusion of fluorescent dye by a side population, retention of the radionucleotide label, etc. Methodologies have also been successfully employed to identify tumorigenic cells within breast cancers. The most important characteristics of stem cells are the capacity for self-renewal and the regulation of the balance between self-renewal and differentiation. In the mammary gland, signaling pathways, such as Hedgehog (Hh), Wnt/β-catenin, and Notch, play a role in embryogenesis and organogenesis and maintenance of tissues in the adult through regulation of the balance between self-renewal and differentiation of stem cells. Breast TAAs include epitopes from proteins, such as carcinoembryonic antigen and NYBR-1, which are involved in tissue differentiation. Targeting BCSCs may be achieved by a number of approaches such as chemotherapy sensitization of BCSCs, differentiating therapy, targeting stem cell elimination, targeting signaling pathways and drug transporters, and inhibition of regulatory pathways involved in self-renewal. Targeting cells which have the potential to metastasize will be an important aspect of the BCSC field as these are the cells that cause the majority of morbidity and mortality from breast cancer.     

Investigations of prostate epithelial stem cells and prostate cancer stem cells

Although both prostate epithelial stem cells and prostate cancer stem cells are implicated in the differentiation of the normal prostate gland and carcinogenesis of prostate cancer, there has, until recently, been little information regarding their biology. This review summarizes the recent advancements in cell biological research including various in vitro culture systems that have offered the characterization and isolation of prostate epithelial stem cells and prostate cancer stem cells. In addition, the stromal niche or microenvironment of stem cells plays an essential role in proliferation and differentiation of normal stem cells. Stroma surrounding cancer cells, which also provide another unique niche, may involve the initiation and development of cancer stem cells. Investigation of stem cells and their microenvironments in the prostate should lead to the elucidation of biological features and the development of novel treatments for prostate cancer.     

Identification,characterization, and biological relevance of prostate cancer stem cells from clinical specimens

Cancer stem cells (CSCs) are a reservoir of tumor cells that exhibit the properties of self-renewal and the ability to re-establish the heterogeneous cell population of a tumor. They appear therapy-resistant and may be the underlying cause of recurrent disease. Using prostate as a model, this review presents the CSC hypothesis and discusses the role of the androgen receptor in CSCs, the methods used for isolating CSCs, and the therapeutic challenges CSCs have for cancer therapy.     

Neuroendocrine peptides in the prostate

Circulating androgens are required for normal growth and maintenance of function of the prostate. However, the prostate also contains neuroendocrine peptides, found either in nerve terminals or in prostatic neuroendocrine cells, which are likely to regulate prostate growth or function. The neuronal peptides are likely to participate in the regulation of the synthesis and secretion of prostatic secretory products. While the function of the neuroendocrine cells is undefined, there is evidence for growth-regulating effects of several neuroendocrine cell peptides. Since neuroendocrine differentiation has been correlated with tumor grade and poor prognosis in prostate cancer, the peptide products of the neuroendocrine cells may influence cancer cell replication as well. Recent evidence in other tissues suggests that peptide hormone receptor second-messenger systems may interact with steroid receptors to modulate their actions. These findings raise the possibility that prostatic neuroendocrine peptides may modulate the response of prostate to androgens.     

The characterization of epithelial and stromal subsets of candidate stem/progenitor cells in the human adult prostate

OBJECTIVES: Questions regarding the cell source and mechanisms in the initiation and progression of prostate cancer are today still open for debate. Indeed, our knowledge regarding prostate cell regulation, self-renewal, and cytodifferentiation is presently rather limited. In this study, we investigated these processes in the normal adult human prostate. METHODS: Dynamic expression patterns in prostate stem/progenitor cells, intermediate/transit-amplifying cells, and cell lineages were immunohistochemically identified in an in situ explant renewal model of the human normal/benign adult prostate (n=6). RESULTS: Cells with a basal phenotype proliferated significantly in explant cultures, whereas luminal cells went into apoptosis. Results further show down-regulation in tissue cultures of the basal and hypothetical stem cell marker Bcl-2 in the majority of cells, except in rare putative epithelial stem cells. Investigation of established (AC133) and novel candidate prostate stem/progenitor markers, including the cell surface receptor tyrosine kinase KIT and its ligand stem cell factor (SCF), showed that these rare epithelial cells are AC133(+)/CD133(low)/Bcl-2(high)/cytokeratin(+)/vimentin(-)/KIT(low)/SCF(low). In addition, we report on a stromal population that expresses the mesenchymal marker vimentin and that is AC133(-)/CD133(high)/Bcl-2(-)/cytokeratin(-)/KIT(high)/SCF(high). CONCLUSIONS: We provide evidence for epithelial renewal in response to tissue culture and for basal and epithelial stem/progenitor cell recruitment leading to an expansion of an intermediate luminal precursor phenotype. Data further suggest that SCF regulates prostate epithelial stem/progenitor cells in an autocrine manner and that all or a subset of the identified novel stromal phenotype represents prostate stromal progenitor cells or interstitial pacemaker cells or both.     

Prostate cell cultures as in vitro models for the study of normal stem cells and cancer stem cells

Current existing therapies for prostate cancer eradicate the majority of cells within a tumor. However, most patients with advanced cancer still progress to androgen-independent metastatic disease that remains essentially incurable by current treatment strategies. Recent evidence has shown that cancer stem cells (CSCs) are a subset of the tumor cells that are responsible for initiating and maintaining the disease. Understanding normal stem cells and CSCs may provide insight into the origin of and new therapeutics for prostate cancer. Normal stem cells and CSCs have been identified in prostate tissue by the use of several markers or techniques. Although research on stem cells has been limited by the lack of suitable in vitro systems, recent studies show that not only primary cells but also several established cell lines may exhibit stem cell properties. This review discusses various in vitro culture systems to propagate normal prostate stem cells and prostate CSCs together with molecular markers. These in vitro cell culture models should be useful for elucidating the differentiation of prostatic epithelium and the biological features of prostate cancer.     

前列腺癌干细胞研究进展

前列腺癌是男性患者中最常见的癌症,严重危害男性的健康。目前关于前列腺癌的发病理论并不能完全解释该疾病异质性、转移性生长、耐药性和肿瘤复发的机制。前列腺干细胞是前列腺中具有自我再生和增殖功能的异质亚群,能产生构成前列腺上皮的所有细胞谱系。根据癌症干细胞假说,前列腺癌可能是一种干细胞疾病。前列腺癌干细胞具有明显的克隆形成性和治疗抵抗性,并在前列腺恶性肿瘤的发生和发展中起决定性作用,选择性靶向这些干细胞的治疗策略已引起了广泛的关注。本文主要论述前列腺癌干细胞的起源、鉴定、分子标记及临床应用,揭示癌症干细胞的生理功能,并获得有关其可能参与前列腺癌发病机理的基本知识,为将来开发有效的治疗策略提供理论基础。     

Epithelial stem cells in human prostate growth and disease

Benign prostatic hyperplasia and prostate cancer arise as a consequence of changes in the balance between cell division and differentiation. Little, however, is known about the control of this process. Stem cells are a small population of cells that divide occasionally to produce transit-amplifying cells that in turn produce the differentiated cell types of the tissue. It is believed that cancer cell proliferation is also driven by stem cells. We have shown that around one in 200 prostate epithelial cells have characteristics of stem cells and that these cells are contained within a population with a distinct keratin expression pattern. Work is now ongoing to identify markers for these cells that will allow us to study the role they play in prostatic disease.     

Androgen Receptor Mediated Growth of Prostate (Cancer)

The understanding of the mode of action of androgens requires insight in the cell biological architecture of the prostate. In the prostate secretory acini, morphologically two cell layers can be discriminated; i.e. the basal and the luminal compartment. The stem cells are thought to be located in the basal compartment. The stem cells have the unique capacity of self-renewal, providing the full repertoire to develop the differentiated ductal system via transient proliferating/amplifying (TP/A) intermediate stem cells. These are in fact early and late progenitors for the exocrine/secretory and neuro-endocrine cells. Exocrine differentiation occurs in the majority of the luminal compartment and is identified by the expression of the androgen receptor (AR), keratin 18 (K18) and prostate specific antigen (PSA). Neuro-endocrine cells are dispersed in the prostate epithelium and express K5 and typical neuro-endocrine markers such as chromogranin A and/or bombesin. The exocrine lineage of differentiation is critically dependent on androgens and thus androgen deprivation therapy will result in declining numbers of secretory/exocrine cells, while the number of stem cells and TP/A intermediate stem cells remains stable. This implies that stem cells and TP/A intermediate stem cells are for their renewal androgen independent, although the TP/A intermediate stem cells are androgen sensitive for expanding the epithelial compartment.Recently, these TP/A intermediate stem cells gained attention because they are thought to play an important role in normal prostate growth as well as in neoplastic transformation. Current hypotheses suggest that from the TP/A cell population, the cancer stem cell develops. Hypothetically, the more committed the cancer stem cell is, the more sensitive the tumour might be for androgen ablation, which could explain why some patients are long-term hormone therapy responders, while others are intrinsically androgen independent. In conclusion, typing of the transformed TP/A intermediate stem cells could enable to discriminate long-term hormone responders from non-responders and could help individualisation of prostate cancer therapy in the future.     

Hedgehog signaling in the prostate

PURPOSE: Recent discoveries highlight the importance of the hedgehog signaling pathway in prostate growth regulation. We reviewed the role of hedgehog signaling in prostate development, adult prostate homeostasis and prostate cancer. MATERIALS AND METHODS: A comprehensive review of all relevant literature was done. RESULTS: Epithelial expression of hedgehog ligand during prostate development exerts autocrine and paracrine signaling activities that regulate growth and differentiation. Hedgehog signaling also occurs in the adult human prostate but to our knowledge the influence on epithelial proliferation and/or differentiation is unknown. Robust hedgehog signaling occurs frequently in prostate cancer, and autocrine and paracrine signaling have been shown to accelerate the growth of xenograft tumors. Autocrine signaling has been implicated in stimulating stem/progenitor cells and increased hedgehog pathway activity may be a characteristic of advanced, androgen independent cancer. The plant alkaloid cyclopamine is a specific chemical inhibitor of hedgehog signaling that produced sustained regression of established xenograft tumors. CONCLUSIONS: Hedgehog signaling has an important role in prostate development and it appears to be a characteristic feature of prostate cancer. It stimulates tumor growth and may exert a specific role in the proliferation of tumor stem cells. The development of hedgehog inhibitors based on the action of cyclopamine holds promise for novel treatments to slow or arrest tumor growth.     

Smart drugs in prostate cancer

OBJECTIVES: Growth signaling is instrumental in tumor development. Insight into signaling pathways by molecular and cellular biology has changed the development of new anticancer agents. Outside the field of urology specifically targeted drugs such as imatinib mesylate and gefitinib showed impressive anticancer activity in chronic myeloid leukemia and non-small cell lung cancer, respectively. METHODS: Literature search of PubMed documented publications and abstracts from meetings. RESULTS: Preclinical data in prostate cancer shows upregulation of a wide variety of growth factors and their receptors such as PDGF, EGF, IGF, FGF, and VEGF suggesting efficacy of agents targeting these pathways. Here the preclinical evidence and first clinical data on the use of growth signal targeting in prostate cancer is reviewed. Although some anticancer efficacy of signal transduction inhibition monotherapy was reported, combination with chemotherapy and radiotherapy seemed most promising in prostate cancer. CONCLUSION: So-called smart drugs are small molecules targeted at specific growth signaling pathways. These new drugs will dominate clinical trials in the years to come either as single-drug modality, but more likely as combination treatment.     

食管鳞状细胞癌干细胞研究的现状及进展

摘要：越来越多的研究表明肿瘤细胞中存在肿瘤干细胞,它与肿瘤的起始、生长、转移及化疗抗性有密切关系。食管鳞癌细胞中也被发现具有干细胞特性的肿瘤细胞,这类细胞具有自我更新、分化潜能、裸鼠成瘤和化疗抗性,这类细胞将在肿瘤靶向治疗中发挥重要的作用。目前培养和分离食管鳞癌干细胞的方法主要有免疫荧光激活细胞分离法、免疫磁珠激活细胞分离法、悬浮培养法、侧群细胞分离法等。本文对当前食管鳞癌干细胞的研究方法、生物学特性及不足进行了综述,并认为食管鳞癌干细胞需要联合多个细胞标记进行研究。     

Effective enrichment of prostate cancer stem cells from spheres in a suspension culture system

BackgroundStem-like prostate cancer cells are also called prostate cancer stem cells (PrCSCs). These rare cells are supposed to be highly tumorigenic and to be involved in maintenance of tumor homeostasis and mediation of tumor metastasis. Methods for sorting PrCSCs are mainly based on sorting cells with the marker (CD133⁺/CD44⁺) or side population cells. However, CD133⁺/CD44⁺ cells or side population cells are very rare or even undetectable. The scarcity of approaches for isolation and purification of PrCSCs is the main obstacle to studying PrCSCs.MethodsIn the present study, suspension culture was used for enrichment of PrCSCs. And PrCSCs were verified by side population technology, drug sensitivity assays, and the molecular marker analysis of prostate cancer stem cell.ResultsPC3 cells survived and formed spheres in nonadherent suspension culture. The percentage of CD44⁺/CD133⁺ cells was 18-fold higher in the nonadherent sphere-forming cell population than in the adherent PC3 cell population (13.94% vs. 0.77%, respectively). This side population was increased to 3.1% in the nonadherent population but undetectable in adherent population. Resistance to cisplatin was higher in the nonadherent cells than adherent cells.ConclusionSuspension culture can be used to enrich for PrCSCs. This approach will aid prostate stem cell biology research and facilitate identification of novel therapeutic agents for prostate cancer.     

Cancer stem cells in prostate cancer

Prostate cancer (P-Ca) remains a leading cause of cancer-related death in men. Lately, increasing evidence for a hierarchically organized cancer stem cell (CSC) model emerged for different tumors entities, including P-Ca. CSCs are defined by several characteristics including self-renewal, pluripotency and tumorigenicity and are thought to be responsible for tumor recurrence, metastasis and cancer related death. In this review we discuss the recent research in the field of CSCs, its limitations and therapeutical implications in general and specifically in P-Ca.     

Hypermethylation of the caveolin-1 gene promoter in prostate cancer

BACKGROUND: Hypermethylation of CpG islands in the promoter regions of tumor suppressor genes is one mechanism of tumorigenesis. Caveolin-1 (Cav-1), a gene coding for the structural component of cellular caveolae, is involved in cell signaling and has been proposed to be a tumor suppressor gene in several malignancies. This gene maps to 7q31.1, a site known to be deleted in some prostate tumors. We chose to examine the methylation status of the promoter region of Cav-1 to determine whether this gene could function as a tumor suppressor in prostate cancer METHODS: Genomic DNA from both tumor and normal prostate epithelial cells was obtained from paraffin-embedded prostate sections by laser capture microdissection (LCM). The methylation status of 24 CpG sites at the 5' promoter region of Cav-1 was analyzed by bisulfite-direct-sequencing after amplification by PCR using primers specific for bisulfate modified DNA. Immunohistochemistry staining with a cav-1-specific antibody was also performed to evaluate the expression of the gene RESULTS: Twenty of the 22 (90.9%) informative cases showed promoter hypermethylation in the tumor cell population when compared with adjacent normal prostate cells with an average Methylation Index (potential frequency of total possible methylated Cs) from tumor cells equal to 0.426 vs. 0.186 for normal cells (P = 0.001). While no association with Gleason grade was found, overall increased methylation correlated with PSA failure (P = 0.016), suggestive of clinical recurrence. Elevated immunoreactivity with a Cav-1 antibody was observed in tumor cells from 7 of 26 prostate samples tested; this was associated with a Gleason score but not correlated with PSA failure or Methylation Index CONCLUSIONS: CpG sites at the 5' promoter of Cav-1 are more methylated in tumor than in adjacent normal prostate cells. Hypermethylation of the Cav-1 promoter supports the notion that Cav-1 may function as a tumor suppressor gene in prostate cancer and evidence is presented suggesting that methylation status of this gene is not only a marker for cancer but also may be predictive of outcome.     

Cellular Quiescence in Mammary Stem Cells and Breast Tumor Stem Cells: Got Testable Hypotheses?

Cellular quiescence is a state of reversible cell cycle arrest and has more recently been shown to be a blockade to differentiation and to correlate with resistance to cancer chemotherapeutics and other xenobiotics; features that are common to adult stem cells and possibly tumor stem cells. The biphasic kinetics of mammary regeneration, coupled to its cyclic endocrine control suggest that mammary stem cells most likely divide during a narrow window of the regenerative cycle and return to a state of quiescence. This would enable them to retain their proliferative capacity, resist differentiation signals and preserve their prolonged life span. There is accumulating evidence that mammary stem cells and other adult stem cells utilize quiescence for this purpose, however the degree to which tumor stem cells do so is largely unknown. The retained proliferative capacity of mammary stem cells likely enables them to accumulate and harbor mutations that lead to breast cancer initiation. However it is currently unclear if these causative lesions lead to defective or deranged quiescence in mammary stem cells. Evidence of such effects could potentially lead to the development of diagnostic systems that monitor mammary stem cell quiescence or activation. Such systems may be useful for the evaluation of patients who are at significant risk of breast cancer. Additionally quiescence has been postulated to contribute to therapeutic resistance and tumor recurrence. This review aims to evaluate what is known about the mechanisms governing cellular quiescence and the role of tumor stem cell quiescence in breast cancer recurrence.     

Neuroendokrine Differenzierung im Prostatakarzinom

Neuroendocrine (NE) differentiation frequently occurs in common prostatic malignancies and has attracted increasing attention in contemporary prostate cancer research. This particular phenotype, however, usually escapes pathological and clinical detection in routine practice. The present review focuses on the biological properties of NE tumor cells that make them resistant to androgen deprivation and radiation therapy. NE cells produce a number of hormonal growth factors (e.g., serotonin) that may act through endocrine, paracrine, and autocrine mechanisms. Morphogenetic studies have identified intermediate phenotypes between the three basic cell types of the prostatic epithelium indicating their common origin from stem cells located in the basal cell layer. Virtually all prostatic adenocarcinomas show NE differentiation as defined by the most commonly used endocrine marker chromogranin A. Clinical studies suggest that the extent of NE differentiation increases with tumor progression and the development of androgen insensitivity. NE differentiation exclusively occurs in the G0 phase of the cell cycle in which tumor cells are usually resistant to radiation therapy and cytotoxic drugs. In addition, NE tumor cells also escape programmed cell death. Even under androgen deprivation, only 0.16% of NE tumor cells show apoptotic activity. This indicates that the vast majority of NE tumor cells represent an immortal cell population in prostate cancer. Although NE tumor cells do not proliferate, they produce a number of NE growth factors with mitogenic properties that maintain cell proliferation in adjacent (exocrine) tumor cells through a paracrine mechanism. NE tumor cells consistently lack the androgen receptor and are androgen insensitive in all stages of the disease. They derive through a process of intermediate differentiation from exocrine tumor cells, the most prevalent phenotype in common prostatic adenocarcinoma. Elevated serum levels of chromogranin A in prostate cancer patients correlate with poor prognosis and are scarcely influenced by either androgen deprivation or chemotherapy. Looking for NE differentiation is recommended in the pathological and clinical evaluation of prostate cancer patients for whom radiation and androgen deprivation are therapeutic options.