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Despite reports of sex steroid receptor and COX2 expression in desmoid-type fibromatosis, responses to single agent therapy with anti-estrogens and non-steroidal anti-inflammatory drugs are unpredictable. Perhaps combination pharmacotherapy might be more effective in desmoid tumors that co-express these targets. Clearly, further understanding of the signaling pathways deregulated in desmoid tumors is essential for the development of targeted molecular therapy. Transforming growth factor–β (TGFβ) and bone morphogenetic proteins (BMP) are important regulators of fibroblast proliferation and matrix deposition, but little is known about the TGFβ superfamily in fibromatosis. A tissue microarray representing 27 desmoid tumors was constructed; 14 samples of healing scar and six samples of normal fibrous tissue were included for comparison. Expression of selected receptors and activated downstream transcription factors of TGFβ family signaling pathways, β–catenin, sex steroid hormone receptors and COX2 were assessed using immunohistochemistry; patterns of co–expression were explored via correlational statistical analyses. In addition to β–catenin, immunoreactivity for phosphorylated SMAD2/3 (indicative of active TGFβ signaling) and COX2 was significantly increased in desmoid tumors compared with healing scar and quiescent fibrous tissue. Low levels of phosphorylated SMAD1/5/8 were detected in only a minority of cases. Transforming growth factor–β receptor type 1 and androgen receptor were expressed in both desmoid tumors and scar, but not in fibrous tissue. Estrogen receptor–β was present in all cases studied. Transforming growth factor–β signaling appears to be activated in desmoid-type fibromatosis and phosphorylated SMAD2/3 and COX2 immunoreactivity might be of diagnostic utility in these tumors. Given the frequency of androgen receptor, estrogen receptor–β and COX2 co-expression in desmoid tumors, further assessment of the efficacy of combination pharmacotherapy using hormonal agonists/antagonists together with COX2 inhibitors should be considered.
Desmoid-type fibromatosis (desmoid tumor) is a fibroblastic-myofibroblastic tumor with a rather unpredictable clinical course.[1-3] Whereas some tumors are slow growing and may regress spontaneously, others grow rapidly and recur repeatedly after complete surgical resection. There are few clinicopathological predictors of disease recurrence. Even the prognostic significance of positive surgical resection margins is controversial.[1, 2, 4, 5] Despite lacking metastatic potential, most desmoids are locally aggressive and cause significant morbidity. Other than wide surgical re-excision and radiation therapy, there is no standard treatment for recurrent disease, a testimony to the limited efficacy of most adjuvant treatment regimens.[2-6]
Expression of estrogen receptor–β and COX2 has been documented in many cases of deep fibromatosis,[7-11] but response rates to single agent anti-estrogen therapies and non-steroidal anti-inflammatory drugs are limited and unpredictable. Co–expression of target proteins might account for responses to combined steroidal and non-steroidal anti-inflammatory drugs in some patients.[12, 13] Since interactions between cell signaling pathways are complex and often redundant, aberrancy in one signaling pathway is often corrected through compensatory (de)regulation of another pathway. This might not only explain why efficacy of single pharmaceutical agents is limited, but also suggests that other pathways might represent additional targets for inhibitor therapy in this disease.[1, 14]
Myofibroblast proliferation, differentiation, migration, angiogenesis and extracellular matrix synthesis during wound healing or fibrosis are processes driven by a variety of paracrine signals, including transforming growth factor–β (TGFβ).[15-18] It is well known that the majority of desmoid tumors harbor mutations of either the β-catenin (CTNNB1) or adenomatous polyposis coli genes of the Wnt signaling pathway, which are thought to deregulate proliferation and invasiveness of fibroblasts.[6, 14, 19] That TGFβ stimulates CTNNB1-mediated transcription suggests that TGFβ might be an important factor in desmoid tumorigenesis.[20-23] Active TGFβ signaling has been reported in desmoid myofibroblasts,[21, 24, 25] and expression of TGFβ-related cytokines such as bone morphogenetic proteins (BMP) has also been described in desmoid tumors.
Current advances in targeted molecular therapy offer new opportunities to treat fibromatosis patients. A better understanding of the signaling pathways active in fibromatosis is necessary to determine the theoretical potential for inhibitor therapy in this debilitating disease. Selected signaling molecules, particularly those of the TGFβ signaling family, were evaluated in 27 cases of deep fibromatosis and compared with healing scar (a reactive myofibroblastic proliferation) and non-neoplastic fibrous tissue. Co-expression patterns were explored using correlational analyses. Although TGFR1 expression levels are similar in desmoid tumors and scars, phosphorylated SMAD2/3 and COX2 levels are significantly greater in desmoid tumors. In contrast, phosphorylated SMAD1/5/8 was detectable in only a minority of desmoids and scar tissues. While estrogen receptor–β expression is present in all desmoids and scar, levels of androgen receptor were variable in these tissues. In addition to being useful diagnostic markers, some of these proteins might be targeted by specific molecular therapeutic agents, offering new treatment regimens that might be potentially individualized to the expression pattern of an individual tumor.
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- Materials and Methods
- Disclosure Statement
The expression of a variety of signaling proteins and activated downstream signal transduction molecules was compared in desmoid tumors, reactive myofibroblasts in healing scars and quiescent fibroblasts in non-proliferative fibrous tissue to identify signal transduction pathways deregulated in desmoid-type fibromatosis. In addition, co–expression patterns were assessed by correlation analysis. Differential patterns of expression of CTNNB1, p–SMAD2/3 and COX2 were observed between desmoid-type fibromatosis, scar and fibrous tissue. Thus, these markers might be useful in distinguishing between desmoid-type fibromatosis and healing scar. The results also suggest that signaling pathways other than the Wnt pathway are abnormally activated in desmoid tumors.
The constituent cell in both desmoid tumor and healing scar tissue is the activated myofibroblast. Therefore, it can be difficult to determine whether a myofibroblastic proliferation is neoplastic or reactive, particularly when evaluating the surgical resection margins of re-excised desmoid tumors. Subtle histological features that are suggestive of a reparative response include relatively small, disordered, irregular fascicles of myofibroblasts, sometimes associated with chronic inflammation, hemosiderin-laden macrophages and areas of neovascularization.
Nuclear CTNNB1 was seen more often in desmoid tumors compared with scar tissue, confirming prior observations.[30, 31] Although nuclear accumulation of CTNNB1 has not been reported in the few examples of non-hypertrophic scars previously studied, it has been demonstrated in myofibroblasts during the proliferative phase of wound healing, in hypertrophic scars and in rare cases of reactive-type fasciitis, suggesting that this signaling pathway is important in the physiological regulation of fibroblast proliferation.[14, 19, 23, 30, 32] Given the occasional presence of nuclear CTNNB1 in non-neoplastic and reactive myofibroblasts, documentation of strong immunoreactivity for p–SMAD2/3 and/or COX2 might be of clinical utility in the differential diagnosis between desmoid-type fibromatosis and these reactive myofibroblastic proliferations in the appropriate clinical setting.
TGFR1 is a serine/threonine kinase receptor that in response to cytokine signaling phosphorylates SMAD2 and SMAD3, which then translocate to the nucleus and regulate the transcription of a number of genes involved in a variety of mesenchymal cell processes, including proliferation, myofibroblastic differentiation, angiogenesis and extracellular matrigenesis.[15, 16, 18, 21, 24, 25, 28] Increased TGFβ activity has been implicated in pathological myofibroblastic proliferations such as keloid formation[33, 34] superficial fibromatoses[35-37] and hypertrophic scar. The TGFβ signaling family is also activated in desmoid tumors.[21, 39, 40] In the present study, TGFR1 staining intensity was similar in both desmoid-type fibromatosis and scar tissue, but it was not detected in quiescent fibrous tissue. In contrast, increased levels of the activated isoform of the TGFR1 downstream signaling molecule p–SMAD2/3 were detected in most desmoid tumors, whereas scar tissue showed low levels in less than one-third of samples. Amini Nik and colleagues previously demonstrated increased levels of p–SMAD2/3 in desmoid tumor cells compared with normal fibrous tissue. Thus, although TGFR1 is present in both desmoid tumor and healing scar tissue, it appears to be activated preferentially (and perhaps aberrantly) in the neoplastic myofibroblasts of desmoid tumors. Despite the reported presence of BMP in desmoid tumors, the BMP receptor signaling pathway does not appear to be activated in desmoid-type fibromatosis or scar tissue, as phosphorylated SMAD1/5/8 was detected in <15% of study cases.
CTNNB1-mediated transcriptional activity can be stimulated by Wnt-independent pathways such as TGFβ signaling.[20, 32] Activation of the TGFβ-receptor pathway has been shown to increase CTNNB1 activity in myofibroblasts during the proliferative phase of wound healing and in desmoid-type fibromatosis in vitro.[21, 22] As both TGFR and atypical Wnt signaling are activated in desmoid tumors, perhaps inhibition of TGFR signaling might be of therapeutic value in desmoid-type fibromatoses despite the absence of correlations between CTNNB1 and TGFR1 or p–SMAD2/3 immunoreactivity seen in the present study.[20-22] In addition to CTNNB1 and p–SMAD2/3, increased levels of COX2 were also more often observed in desmoid tumors than in scar tissues, similar to previous findings.[10, 27] COX2 expression is induced by numerous cytokines and growth factors and is quite common in both inflammatory and neoplastic conditions. Like TGFβ, COX2-mediated prostaglandin signaling might also stimulate CTNNB1-dependent transcription and cell proliferation,[42-44] as inhibition of COX2 reduces proliferation of desmoid tumor cells in vitro.  Correlation analysis performed in the present study demonstrated associations between TGFR1, p–SMAD2/3 and COX2 expression, suggesting that TGFβ activity increases COX2 expression in desmoid tumors, as in other cell types.[41, 46]
Variable levels of androgen receptor expression were present in desmoids and scars, which were significantly higher than in fibrous tissue. The reported prevalence of androgen receptor expression in fibromatosis ranges from <10% to 100%, depending on the methodology used.[11, 47, 48] As shown previously, estrogen receptor–α and progesterone receptor are not detectable in fibromatosis, while expression of estrogen receptor–β is ubiquitous in fibrous and myofibroblastic tissues.[7-9, 11] Similar to previous studies, no associations were observed between sex steroid receptor expression and patient age or sex. There also appears to be no correlation between sex steroid receptor status and response to hormonal therapy.
The androgen receptor modulates Wnt signaling, possibly by binding CTNNB1-related transcriptional complexes in a cell type-specific manner.[49-51] Androgen receptor also appears to inhibit COX2 expression and modulate TGFβ signaling independent of androgen ligands.[52, 53] In the present study, androgen receptor levels were found to correlate with both p–SMAD2/3 and COX2 in desmoid-type fibromatosis. As the androgen receptor can regulate Wnt and TGFβ signaling as well as COX2 expression, perhaps androgen manipulation therapy would be beneficial to fibromatosis patients. Supporting this suggestion is recent evidence from Hong and colleagues, who showed that testosterone increased cell proliferation and CTNNB1 levels in cultured desmoid tumor cells. The frequent co–expression of androgen receptor, COX2 and estrogen receptor–β suggests that specific pharmacotherapeutic agents used in combination might show superior efficacy than single agents alone, particularly when tailored to the expression profile of an individual tumor.
Aberrations of the Wnt signaling pathway are well documented in desmoid-type fibromatosis. In the present study, we showed that TGFβ activity and COX2 expression are also increased in desmoid tumors compared with non-neoplastic myofibroblasts and might be useful ancillary diagnostic markers for desmoid-type fibromatosis. Other experimental evidence of positive interactions between TGFβ and Wnt signaling pathways suggest that the former might be an important factor in the pathogenesis of desmoid tumors, as in other tumor types.[20-22] These interactions might provide additional molecular targets for systemic therapy of intractable desmoid tumors. As signaling molecules such as estrogen receptor–β, androgen receptor and COX2 are frequently co-expressed in desmoid-type fibromatosis, specific pharmacotherapeutic agents used in combination might show superior efficacy than single agents alone. Consideration should be given to combination therapy based on the specific expression profile of an individual tumor, because the complex cross-signaling between these pathways intimate that combined therapy might result in more robust treatment responses.