Cancer Stem Cells and Microenvironment in Prostate Cancer Progression
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- Liao, CP., Adisetiyo, H., Liang, M. et al. HORM CANC (2010) 1: 297. doi:10.1007/s12672-010-0051-5
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For a study of interactions between the cancer-associated fibroblasts (CAFs) and the putative prostate cancer stem cells (CSCs), we used a conditional Pten deletion mouse model of prostatic adenocarcinoma to isolate both CAF cultures and CSC-enriched cell fractions from the primary tumors. The CSC subpopulation exhibited a collective phenotype of Lin−/SCA-1hi/CD49fhi/p63hi/CK5hi/ARlo/CK18lo/Survivinhi/Runx2hi and contained cells with the ability to both self-renew and differentiate into basal and luminal cells in vitro. The spheroids generated from the CSC-enriched subpopulation mimicked the glandular structures that could be produced from a similarly isolated cell fraction from the normal mouse prostate. The efficiency of spheroid formation was found to be influenced differentially by the nature of the fibroblasts that were co-cultured in the 3-D system. The growth and differentiation properties of the CSCs were significantly more enhanced by factors released from CAFs relative to normal prostate fibroblasts (NPFs). Additionally, increased commitment to differentiation to the luminal cell lineage was noted when CAFs were present. When CSCs admixed with either CAFs or NPFs were examined for formation of prostatic glandular structures in renal grafts in vivo, the lesions formed were generally more in numbers in the presence of CAFs than NPFs. Furthermore, lesions formed with CAFs often displayed tumor-like complex histopathology and contained increased numbers of proliferating cells. Taken together, the results suggested that the CAFs in the prostate tumor microenvironment can contribute to the biologic properties of the CSCs and by this account may play a major role in prostate tumorigenesis and progression. Thus, it would be important now to identify the paracrine and/or juxtacrine factors that are responsible for the stimulation of the cancer stem cells.
KeywordsProstate cancer stem cells Cancer-associated fibroblasts Prostate cancer Mouse models of prostate cancer Heterotypic cell-cell interactions in prostate cancer
It has been proposed that cancer contains a minor population of cells that can self-renew while simultaneously giving rise to tumor cells [1, 2]. Such cells may also mediate tumor homeostasis, progression, metastasis, and recurrence. These cells have been referred to as tumorigenic cells, tumor-initiating cells, or cancer stem cells (CSCs). Compared to CSCs, other tumor cells posses only limited proliferation and tumorigenic potentials. CSCs, first described in tumors of the hematopoietic system, were found to share certain cell surface marker phenotype with the normal hematopoietic stem cells . In recent years, presence of a similar relationship between tissue stem cells and CSC fractions of solid tumors from various organs including the prostate is also being increasingly implicated [3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14].
All carcinoma tissues, in general, consist of malignant epithelial cells and their progenitors, a variety of stromal cells including fibroblasts (and myofibroblasts), endothelial cells, pericytes, and inflammatory cells. Extracellular proteins secreted by these different cell types are present in the tumor microenvironment. These constitute a complex array of growth factors, cytokines, chemokines, and adhesive molecules that are likely to alter the balance between proliferation, survival, differentiation, and quiescence in both cancer cell populations and their progenitors during tumor progression, metastasis, or recurrence. The important contribution of stroma to genesis and progression of a variety of tumors has been described [15, 16, 17, 18, 19]. Myofibroblasts, characterized by their expression of α-smooth muscle actin (α-SMA), which represent a large fraction of stromal fibroblastic cell populations in cancers, particularly in prostate and breast cancers, are also present in areas of wound healing and chronic inflammation. The myofibroblasts are known to stimulate epithelial cell growth through their ability to produce extracellular matrix and by secretion of growth factors and cytokines and to support angiogenesis [16, 17, 20].
There is evidence that myofibroblastic cells from prostate tumors, termed as “cancer-associated” fibroblasts (CAFs) can enhance the tumorigenic potential of the epithelial compartment [21, 22]. In this regard, an important new question is if fibroblasts acquire genetic and/or epigenetic alterations during tumor progression. Recently in a mouse model of prostate cancer, it has been demonstrated that a selective evolution of stromal mesenchyme does occur with p53 loss in response to epithelial tumorigenesis . Such selection of aberrant stromal cells in some human epithelial cancers was earlier implicated in the cancer progression [24, 25, 26, 27, 28], although lack of detection of changes was also reported . Inflammation and innate immunity that occur in the microenvironment are also widely recognized to play important roles in epithelial cancer progression [19, 30].
The mouse prostate has been the primary focus of research in regards to normal stem cells in the animal prostate. There are three epithelial cell types from which the prostate is composed: luminal cells, the differentiated secretory cells which are the major cell type; neuroendocrine cells, rare and morphologically heterogeneous cells present in both the basal and luminal cell layers; and basal cells, the undifferentiated, non-secretory cells that may contain the progenitor cells of the prostate [31, 32]. The proximal region attached to the urethra is the probable location of stem cells because it contains a high number of label-retaining cells, which express stem cell-specific markers, are resistant to androgen ablation, and have a greater capacity to regenerate prostate tissue in growth assays as compared to cells located in the distal regions [33, 34]. Stem cells reside in a specialized microenvironment, in which their quiescence and proliferation appear to be regulated by factors such as transforming growth factor-β, epidermal growth factor, insulin-like growth factor, etc. [32, 35, 36]. There is increasing evidence that cell markers such as stem cell antigen-1 (SCA-1), laminin receptor α6 integrin (CD49f), CD133 (prominin), CD44, and CD117 (c-kit, stem cell factor receptor) can be used to enrich for stem cells of the mouse prostate [32, 37, 38]. Interestingly, there is evidence in a mouse xenograft model that the α6 integrin, whose expression is upregulated in prostate cancer cells , can be converted to a novel form by cleavage of the laminin-binding domain from the cell surface by urokinase-type plasminogen activator, and such cleavage may permit extravasation of human prostate cancer cells from the circulation to bone [40, 41]. Another exciting new evidence is that a single mouse prostate stem cell defined by a SCA-1+ CD133+ CD44+ CD117+ phenotype and implanted under the renal capsule can generate secretion-producing prostatic ducts consisting of basal, luminal, and neuroendocrine cells .
In regard to modeling human prostate cancer in mice, transgenic mice with activation or deficiency of genes such as androgen receptor, fibroblast growth factor 8, c-Myc, Akt, Pten, Nkx3.1, Rb, p53, Apc, or some of their combinations demonstrate gene-dependent development of the various stages of prostate cancer [42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54]. Preliminary findings suggest that different genetic events have unequal transforming effects on stem cells and their progeny . One mouse model  that closely recapitulates the human prostate cancer is the conditional Pten deletion mouse line (cPten−/−), which concerns prostate epithelium-specific loss of PTEN, a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway. In humans, approximately 70% of primary prostate cancers exhibit a loss of at least one Pten allele, and functional suppression of both alleles is associated with advanced disease [56, 57]. In mice, Pten deletion of one allele leads to the development of high-grade prostate intracellular neoplasia , but loss of both alleles results in adenocarcinoma at age beginning from 9 weeks and invasive prostate cancer that metastasizes to primarily to lymph nodes subsequently . The mouse models present the potential to increase our understanding of the relevance of various cellular components and signaling proteins that occur in the tumor microenvironment to the biology of prostate cancer stem cells in the progression and recurrence of the disease.
Relevant Characteristics of the Tumor Model and Tumor-Derived Fibroblast Cultures
Properties of the Tumor-Derived Cancer Stem Cell Subpopulation
SCA-1 is a marker that has been demonstrated to enrich for stem cells in normal mouse hematopoietic, skin, cardiac, and testicular tissues [64, 65, 66, 67] and prostate epithelium . In normal murine prostate, androgen deprivation leads to enrichment of SCA-1+ cell subpopulation in the proximal region, which is considered to be a niche for the resident stem cells . There is evidence that Pten deletion may cause expansion of SCA-1+ cell subpopulation in the prostate tumors . Recognizing that SCA-1+ cell fraction isolated from normal mouse prostate can self-renew and differentiate in vitro  and can regenerate prostate glandular structures in vivo , we applied a similar approach in attempts to isolate putative cancer stem cell subpopulation that might be present in the adenocarcinoma of cPten−/−L mice. A recent study also describes the utility of the SCA-1 marker for the enrichment of tumor stem/progenitor cells from the cPten−/− tumors of the prostate .
Detection of prostate glandular structures in grafts formed from CSCs
Primary stromal cells
There is currently a high level of interest in tumor microenvironment with the emphasis placed on the host stroma in tumorigenesis and responses to treatment. To delineate mechanisms of tumor–stroma interactions in cancer, it may be necessary for practical considerations to focus on one stromal cellular compartment at a time. With the accumulation of information on each type of interactions, it may then be possible to deduce a more comprehensive understanding of the role of the stroma on the growth and progression of a specific cancer type. In this report, we describe a model system to investigate the influence of the myofibroblasts present in the prostate tumors on the potency of the putative CSCs from the same tumors. The emphasis is placed on CSCs, which constitute only a minor population because they may be central to homeostasis, progression, and recurrence of the tumors. For this initial work , myofibroblastic CAFs and the CSC were derived from the primary tumors of the conditional Pten deletion model of the mouse prostate adenocarcinoma.
We refined a procedure for the isolation of epithelial cell fractions retaining a small number of cells with properties of putative CSCs from the prostate tumor mouse model. A cell fraction from this tumor model is shown to possess self-renewal and spheroid-forming abilities along with multi-potentiality for differentiation in vitro and the ability to form tumor-like glandular structures in vivo under appropriate conditions. We have observed a significant difference in the pattern of relative expression of certain relevant genes in the subpopulation of epithelial cells with which the functional CSCs segregate. The expressions of basal cell markers CK5 and p63 are strong in CSCs, while those of CK18 and AR are low. The same general pattern is found in the respective subpopulation from the normal mouse prostate. A high level of expression is also noted for two cancer-related genes: Survivin and Runx2 in CSCs. This finding is especially interesting since we have documented a strong correlation between increased levels of Survivin and Runx2 with the growth of the tumor in the model . Moreover, Runx2 appears to be a major regulator of Survivin gene transcription in prostate cancer cells .
We demonstrate that the in vitro spheroid-forming efficiency of the CSC-enriched cells is differentially influenced by the fibroblasts in co-cultures. The modified spheroid-forming co-culture system we used has the promise to be a powerful method to facilitate the studies of paracrine signaling in interactions between stromal fibroblasts and CSCs. As compared to UGSM or NPFs, CAFs enhance spheroid formation by two-fold. It also appears that kinetics of differentiation toward the luminal cell phenotype over basal cell type is favored in the presences of CAFs relative to the other fibroblastic cells. In vivo, the grafts grown from CSCs are found to contain multiple glandular structures in each case, although grafts formed with CAFs appear to exhibit higher proliferative index and increased number of lesions with more complex morphology as compared to those formed with NPFs. These observations with CAFs underscore a significant role of CAFs in CSC biology and motivate studies to uncover the responsible molecular interactions. It is likely that many different molecules with positive or opposite effects may act in concert to modulate the complex processes of CSC biology. Thus, analysis of each implicated factor alone and in specific combinations will be needed to dissect parameters that converge to drive the cascade to sustain or stimulate CSC properties. Identification of pertinent secreted proteins from the model system will then lead to the scrutiny of analogous factors on human prostate CSCs and their distribution in the stromal compartments in prostate cancer specimens from different stages of the disease.
It is expected that with a better understanding of the signaling between CSCs and CAFs, pathway-specific inhibitors that block CAF–CSC interactions could be designed and tested on the preclinical model systems to better inform clinical development. Ultimate goal of these studies would be to attack prostate cancer at its core, which is the persistence and increased virulence of CSCs with the disease progression. Moreover, a focus on stromal fibroblasts that regulate prostate CSC activity offers a new direction for therapeutic development against a cellular compartment different from the malignant epithelium.
Evidence is presented by using a prostatic adenocarcinoma mouse model to support the notion that, like stem cells of the normal mouse prostate gland, a minor population of epithelial cells exists in the prostate cancer tissue that harbors the potential for both self-renewal and multi-potential differentiation activity. This cellular subpopulation, tentatively designated as cancer stem cells, may indeed be a renewable source for cancer persistence, growth, and re-growth or recurrence. It is also described that both the cell biology and the abnormal behavior of these stem cell-like cells can be robustly influenced by the myofibroblasts that occur in the tumor microenvironment. The characterization of the factors involved in the implicated heterotypic cell–cell interactions may provide new insights to strategies to improve prostate cancer treatment by targeting not only the epithelial compartment but also a fibroblastic compartment of the tumor microenvironment.
This work was supported by the National Institutes of Health research grants RO1 CA59705 and RO1 CA113392 to P. Roy-Burman and, in part, by supports to C-P. Liao and H. Adisetiyo from the California Institute for Regenerative Medicine training grant T1-004.
Conflict of Interest
The authors declare that they have no conflict of interest.