We found that the BMP antagonist GREMLIN 1 is frequently expressedby stromal cells in the microenvironment of human carcinomas,including BCC, and can enhance cell expansion and block differentiationin vitro. Carcinomas are histologically complex tissues comprisingnot only tumor cells but also fibroblasts, smooth muscle cells,endothelial cells, adipocytes, and leukocytes, as well as componentsof the extracellular matrix. Interactions with these cells andfactors in the tumor microenvironment, or tumor cell niche,may also play a critical role in the initiation and progressionof cancer (15, 16, 30). We used a genomic approach to identifyfactors differentially expressed by BCC-associated fibroblastscompared with their nontumor-associated counterparts. Globalgene expression profiling of these two cell populations revealedintrinsic, systematic differences in gene expression programs.We chose to focus on one gene in particular, the BMP antagonistGREMLIN 1. In many settings, BMPs promote differentiation ofstem cells, thus promoting exit from the stem cell compartment(23, 31). These observations led us to hypothesize that BMPantagonists may define a niche for a self-renewing populationin some cancers.
In this model, the role of BMPs and their antagonists in regulatinga self-renewing tumor cell compartment parallels their rolein regulating the normal stem cell compartment (Fig. 5). Innormal physiology, factors (including the BMP antagonists) thatsupport "stemness" of stem cells are often provided by a stemcell "niche," a molecular microenvironment defined by a localizedpopulation of cells that regulates the size of the stem cellcompartment (32, 33). Our data suggest a directly analogousmodel for the tumor context in which the tumor cells requireBMP antagonists coming from the tumor fibroblasts (another specializedstromal compartment) to maintain their expansion.
Our results represent a dramatic example of the differencesbetween stromal cells in cancer and those in the normal tissuecounterpart. Elevated expression of GREMLIN 1 has previouslybeen documented in a small subset of cells in normal skin, theputative epithelial stem cells, compared with other normal skinepithelial cells (34). In our study, GREMLIN 1 RNA was expressedin stromal cells of nearly all BCC samples examined, but undetectablein the vast majority of normal skin sites. The cells that expressGREMLIN 1 have the appearance and immunohistochemical characteristicsof fibroblasts and not cells of epithelial, lymphocytic, endothelial,smooth muscle, or glial origin.
How is this distinct, specialized tumor stromal compartmentinitially established? Does the gremlin 1-rich tumor niche developin response to signals derived from the tumor? If so, the presenceof gremlin 1-expressing fibroblasts could be the product ofeither de novo differentiation, recruitment from a distant site,or preferential expansion of an otherwise rare population inresponse to molecular signals from the tumor cells. In an alternativemodel, the chronology is reversed, that is, a specialized nichefavorable to tumor initiation and expansion may be establishedbefore the tumor can form, perhaps as a result of clonal expansionof a mutant or epigenetically modified clone of fibroblasts.Indeed, the familiar focal, patchy alterations in skin pigmentationand texture, hair morphology, and vascularization seen in aging,sun-exposed skin are consistent with preexisting local clonalfields of altered cells (35). Whatever events lead to the accumulationof gremlin 1-expressing fibroblasts in diverse carcinomas, theability of tumor-derived fibroblasts to maintain this distinctiveexpression program even after many generations of culture exvivo, away from the influence of their tumor counterpart, suggeststhis maintenance is specified by a stable genetic or epigeneticprogram.
The addition of gremlin 1 alone to basal media was not enoughto sustain long-term culture of BCC-derived cells. Thus, futurework is needed to define additional supporting factors presentin the tumor cell niche. As a preliminary step, we have usedRT-PCR to examine the expression of a number of other reportedBMP antagonists, including TSG1, FOLLISTATIN, NOGGIN, and CHORDIN,in whole tissue samples of human BCC and matched nontumor tissue.Like GREMLIN 1, both TSG1 and CHORDIN were typically expressedat higher levels in tumors compared with nontumor controls (Fig.9, which is published as supporting information on the PNASweb site). Further characterization of other factors in thetumor cell niche, combined with the identification of signalsderived from basal cell tumor cells, will help elucidate thereciprocal crosstalk that occurs between the tumor and its microenvironment.Along with GREMLIN 1, other genes that were elevated in BCCtumor-associated fibroblasts included a number of componentsof the Wnt signaling pathway, such as DICKKOPF HOMOLOG 1 (DKK1),a secreted protein inhibitor of the Wnt signaling pathway. TheWnt proteins (along with BMPs) are targets of the Sonic Hedgehogpathway (36). In one report of Wnt pathway activity in BCC,the pattern of nuclear b-catenin showed increased staining atthe periphery of tumor nests, as well as some staining in tumor-adjacentfibroblasts (37). Additional experiments will be useful in uncoveringthe connections between Wnt, Sonic Hedgehog, and BMP signalingin BCC.
We have shown that BMP inhibits expansion of BCC cells in culture,and that gremlin 1 can overcome this inhibition. The mechanismof gremlin 1/BMP action and downstream signaling events, however,remains unclear. Although we have not definitively addressedwhether the effects of gremlin are mediated exclusively throughthe BMP pathway, our data on cultured cells from BCC tumorssuggest that this is likely, because gremlin 1 had no appreciableeffect on cell expansion unless BMP was present. Although wedid observe BMP in some of the tumor cells in vivo, the tumorcells in vitro showed a response to gremlin 1 only in the presenceof exogenous BMP. Thus, the level of BMP production by the culturedtumor cells was not high enough to produce a clear effect atthe plated cell density, possibly because of the effects ofdilution by the media or by loss of normal cell–cell interactionnormally seen in vivo.
The expression of GREMLIN 1 by stromal cells in diverse humancarcinomas, in contrast to its rare expression in correspondingnormal tissues, suggests that expression by cells in the tumormicroenvironment of factors that regulate the self renewal ofthe tumor cells may be a general feature of human cancer. Inhibitingthese critical molecular signals from the tumor microenvironmentmay thus be a useful therapeutic strategy. The potential parallelsbetween stem cell–microenvironment interactions in normaldevelopment and cancer should provide fertile ground for furtherinvestigations.
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