Using Stem Cells to Challenge the Traditional View of the Origin of Tumor Cells
Current Research from the Hernando Lab
Traditionally, mature cells in specific tissues and organs have been regarded as the cell-of-origin of the corresponding tumors. However, the observation that tumor cells need to accumulate genetic and phenotypic alterations over extended time periods has turned the view in recent years to stem cells or progenitors with a prolonged lifespan, broadly distributed in local reservoirs [1]. These cells, in charge of maintaining tissue homeostasis, are now considered as the potential target of neoplastic transformation.
Although this notion has been largely accepted for certain leukemias and lymphomas in which the corresponding hematopoietic progenitors are precisely defined, this theory has not yet permeated the field of solid tumors, with few exceptions [2-4]. The identification of maturation stages in non-hematopoietic (i.e., epithelial, mesenchymal) cell types is proving difficult due to the lack of distinctive markers and the low abundance of such intermediates in adult tissues, except after trauma-induced regeneration.
Our laboratory is studying whether certain sarcomas originate from mesenchymal progenitors [5, 6] and whether melanomas result from transformation of melanocytic precursors [7, 8]. Moreover, we hypothesize that alterations in the normal differentiation process of these progenitors act at early stages of tumor initiation, and that the retention or reactivation of stem cell properties may contribute to tumor progression and aggressive behavior (resistance to therapy, metastasis). A limitation for these studies is our partial understanding of the normal differentiation process of these two lineages.
Fig. 1: A. Phase contrast images of a smooth-muscle (SMC) differentiation time course from mesenchymal stem cells (hMSCs). Characteristic hills (orange arrows) and valleys (yellow arrows) are seen by day 21. (d= number of days post administration of SMDM) B. Immunofluorescence (IF) for MSC marker CD105. C. IF for ASMA (alpha-Smooth Muscle Actin. Left panels (on B and C) correspond to the isotypic controls for each antibody (Neg). D. RT-PCR analysis of SM (SM-MHC) and MSC markers (CD105, CD29 and CD73) during normal SM differentiation. SMC: RNA from smooth muscle cells used as control.
Sarcoma studies
To overcome this limitation, our laboratory is characterizing the differentiation of human Mesenchymal Stem Cells (hMSCs) into smooth-muscle (SMC) (Fig. 1), the lineage of origin of Leiomyosarcomas (LMS), tumors that appear in the uterus, the retroperitoneum and the extremities. In particular, we are focusing on miRNAs, small non-coding RNAs with a critical role in development and tissue specification, as master regulators of smooth-muscle differentiation and thus potential markers of the LMS cell-of-origin. Certain miRNAs up-regulated during hMSC differentiation have been found expressed at low levels in uterine LMS compared to normal myometrium (Danielson and Hernando, unpublished data). We are currently determining whether these candidate miRNAs play an active role in SM differentiation in vitro and in vivo and whether their alteration may lead to a blockade in SM maturation, increased proliferation and/or MSC transformation.
In addition, we have developed a leiomyosarcoma mouse model based on the inactivation of Pten in an early smooth-muscle progenitor (using the transgelin promoter, Tgln) [9]. MSCs isolated from Tgln-cre/Ptenlox/lox mice are being analyzed for the effect of Pten inactivation in stem cell maintenance, proliferation and differentiation. Mice heterozygous for Pten and p53 in the SM lineage develop very aggressive LMS that are able to metastasize to the liver (Guijarro and Hernando, unpublished data). We are currently using these mice tumors to refine the criteria for the isolation of cell subpopulations with increased tumor initiating potential or cancer stem cells (CSCs), and therefore, potentially responsible for leading tumor expansion. For that, we are using different criteria, such as cell sorting based on MSC markers -CD105, CD106, CD73- and stem cell properties -dye exclusion, aldehyde dehydrogenase activity. We are confident that these studies in murine sarcomas will inform our search for similar CSCs in human sarcomas.
Melanoma studies
Melanomas also show phenotypic heterogeneity both in vivo and in vitro, suggesting an origin from a cell with multilineage differentiation abilities. Moreover, malignant melanoma seems to evoke the migratory nature of neural crest and melanoblasts from which melanocytes arise, suggesting that transformation may occur in a melanocytic stem cell or progenitor [10]. Melanoma cells retain their morphologic and biological plasticity despite repeated cloning [11] and express developmental genes. Indirect evidence also supports the presence of melanoma stem-like cells [12] which possess self-renewal capacity, high tumorigenicity, and differentiation into various lineages, including neural, mesenchymal and endothelial, in addition to melanocytic cells [reviewed in [13]).
Our lab has found a miRNA cluster (miR-182-96-183), located in a genomic region frequently amplified in melanoma (7q31-34), as overexpressed in melanoma tissues and cell lines. A member of this cluster, miR-182, controls MITF, a transcription factor with a critical role in melanocytic differentiation [14]. We have demonstrated that upregulation of a member of this cluster, miR-182, promotes migration in vitro and metastasis in vivo (Fig. 2) [14]. Interestingly, miR-182 targets MITF, a transcription factor with a critical role in melanocytic differentiation. Ongoing work using stem cells, melanoma cell lines, and mouse models should unravel the mechanism(s) by which this miRNA contributes to melanoma progression.
Determining the cell-of-origin of sarcomas and melanomas would provide critical insights into the biological and clinical behavior of these tumors. Demonstrating that these tumors contain cells retaining characteristics of progenitors may explain the chemoresistant, invasive and adaptive behavior of these tumors while providing new molecular targets for therapeutic intervention.
Fig. 2: In vivo metastasis assay with B16F10 mouse melanoma cells injected through the lateral tail vein of C57BL/6J mice (n=6). Left: Macroscopic pictures of mice lungs 10 days post-inoculation, and H&E-stained sections of lung metastases (magnification 20x). Right: Quantification of large lung metastases.
References
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