Kristy L. Weber

TGF‐β Promotes the Establishment of Renal Cell Carcinoma Bone Metastasis

Bone metastases develop in ˜30% of patients with RCC, and the mechanisms responsible for this phenomenon are unknown. We found that TGF‐β1 stimulation of RCC bone metastasis cells promotes tumor growth and bone destruction possibly by stimulating paracrine interactions between tumor cells and the bone.

MMP-13 is over-expressed in renal cell carcinoma bone metastasis and is induced by TGF-β1

Bone metastasis occurs frequently in renal cell carcinoma (RCC) patients causing significant morbidity by stimulating excessive osteolysis, yet the mechanisms responsible have been little studied. Matrix metalloproteinases (MMPs) are over-expressed in many cancer types and are believed to play a role in bone metastasis, however, the expression of MMPs in RCC bone metastasis (RBM) has not been investigated. Due to their ability to degrade the main component of organic bone matrix, type I collagen, we investigated the expression of MMP-1, -2, -8, -9, and -13 in RBM. By quantitative (q)RT-PCR, expression of MMP-13 was significantly increased in RBM tissues relative to that in RCC and adjacent normal kidney while no differences in the expression of MMP-1, -2, -8, or -9 mRNA were observed. Correspondingly, increased expression of MMP-13 protein was also observed in RBM relative to RCC by immunohistochemical analysis. Intriguingly, the expression of MMP-13 in the human RBM cell line RBM1-IT4 was stimulated by TGF-β1, a growth factor abundant in the bone microenvironment and known to promote RBM-induced osteolysis in animals. Exposure of RBM1-IT4 cells to TGF-β1 increased MMP-13 mRNA levels as well as the latent and active forms of MMP-13 protein. Further, stable expression of a dominant-negative TGF-β type II receptor in RBM1-IT4 cells inhibited MMP-13 expression following TGF-β1 exposure. These data suggest that MMP-13 expression is elevated in RBM relative to primary RCC and adjacent normal kidney, and is regulated at the cellular level by TGF-β1.

Epithelioid sarcoma: one institution’s experience with a rare sarcoma

BACKGROUNDnEpithelioid sarcomas (ES) are extremely rare soft tissue sarcomas. As such, their clinical behavior and response to treatment are poorly described in the literature.nnnMETHODSnWe queried the centralized cancer registry and pathology archives at the Johns Hopkins Medical Institution and identified 22 patients with a diagnosis of ES. We excluded two patients because of inadequate data. A pathologist reviewed patient charts and reexamined available histological slides. This study was performed with institutional review board approval.nnnRESULTSnThe median age at diagnosis was 27.8 y; most patients (75%) were male. Regional lymph node metastases were present in 10% of patients at presentation. The majority of tumors (57.9%) recurred and 35% recurred more than once, although the number of recurrences did not affect survival (P = 0.48). Patients did not experience a decrease in time to recurrence with increasing number of resections. The median time between resection and recurrence was 1.23 y and the maximum was 18.8 y. Median overall survival was 56.2 mo and 5-y survival was 92%.nnnCONCLUSIONSnOur study reveals that ES is an extremely rare tumor with a protracted and recurrent course, but overall survival may be more favorable than in the past. Patients benefit from aggressive and repeated resection. Epithelioid sarcoma is unique because it metastasizes to regional nodal basins. Extended surveillance is indicated, because recurrences can appear after decades of quiescence.

Identification of prospective factors promoting osteotropism in breast cancer: a potential role for CITED2

Breast cancer metastases develop in the bone more frequently than any other site and are a common cause of morbidity in the form of bone pain, pathological fractures, nerve compression and life‐threatening hypercalcemia. Despite ongoing research efforts, the molecular and cellular mechanisms that regulate breast cancer cell homing to and colonization of the bone as well as resultant pathological bone alteration remain poorly understood. To identify key mediators promoting breast cancer metastasis to bone, we utilized an immunocompetent, syngeneic murine model of breast cancer metastasis employing the mammary tumor cell line NT2.5. Following intracardiac injection of NT2.5 cells in neu‐N mice, metastases developed in the bone, liver and lung, closely mimicking the anatomical distribution of metastases in patients with breast cancer. Using an in vivo selection process, we established NT2.5 sublines demonstrating an enhanced ability to colonize the bone and liver. Genome‐wide cDNA microarray analysis comparing gene expression between parental NT2.5 cells and established sublines revealed both known and novel mediators of bone metastasis and osteolysis, including the transcriptional co‐activator CITED2. In further studies, we found that expression of CITED2 was elevated in human primary breast tumors and bone metastasis compared to normal mammary epithelium and was highest in breast cancer cell lines that cause osteolytic bone metastasis in animal models. In addition, reducing CITED2 expression in NT2.5 cells inhibited the establishment of bone metastasis and osteolysis in vivo, suggesting a potential role for CITED2 in promoting breast cancer bone metastasis.

Enpp1: A Potential Facilitator of Breast Cancer Bone Metastasis

Bone is the most common site of breast cancer metastasis and once established, it is frequently incurable. Critical to our ability to prevent and treat bone metastasis is the identification of the key factors mediating its establishment and understanding their biological function. To address this issue we previously carried out an in vivo selection process to isolate murine mammary tumor sublines possessing an enhanced ability to colonize the bone. A comparison of gene expression between parental cells and sublines by genome-wide cDNA microarray analysis revealed several potential mediators of bone metastasis, including the pyrophosphate-generating ectoenzyme Enpp1. By qRT-PCR and Western analysis we found that expression of Enpp1 was elevated in human breast cancer cell lines known to produce bone metastasis in animal models compared to non-metastatic and normal mammary epithelial cell lines. Further, in clinical specimens, levels of Enpp1 were significantly elevated in human primary breast tumors relative to normal mammary epithelium, with highest levels observed in breast-bone metastasis as determined by qRT-PCR and immunohistochemical analysis. To examine the potential role of Enpp1 in the development of bone metastasis, Enpp1 expression was stably increased in the breast cancer cell line MDA-MB-231 and the ability to colonize the bone following intracardiac and direct intratibial injection of athymic nude mice was determined. By both routes of administration, increased expression of Enpp1 enhanced the ability of MDA-MB-231 cells to form tumors in the bone relative to cells expressing vector alone, as determined by digital radiography and histological analysis. Taken together, these data suggest a potential role for Enpp1 in the development of breast cancer bone metastasis.

CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells.

Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator of ER in breast cancer cells and that its increased expression in tumors may result in estrogen-independent ER activation, thereby reducing estrogen dependence and response to anti-estrogen therapy.

CCL20/CCR6 Signaling Regulates Bone Mass Accrual in Mice.

CCL20 is a member of the macrophage inflammatory protein family and is reported to signal monogamously through the receptor CCR6. Although studies have identified the genomic locations of both Ccl20 and Ccr6 as regions important for bone quality, the role of CCL20/CCR6 signaling in regulating bone mass is unknown. By micro–computed tomography (μCT) and histomorphometric analysis, we show that global loss of Ccr6 in mice significantly decreases trabecular bone mass coincident with reduced osteoblast numbers. Notably, CCL20 and CCR6 were co‐expressed in osteoblast progenitors and levels increased during osteoblast differentiation, indicating the potential of CCL20/CCR6 signaling to influence osteoblasts through both autocrine and paracrine actions. With respect to autocrine effects, CCR6 was found to act as a functional G protein–coupled receptor in osteoblasts and although its loss did not appear to affect the number or proliferation rate of osteoblast progenitors, differentiation was significantly inhibited as evidenced by delays in osteoblast marker gene expression, alkaline phosphatase activity, and mineralization. In addition, CCL20 promoted osteoblast survival concordant with activation of the PI3K‐AKT pathway. Beyond these potential autocrine effects, osteoblast‐derived CCL20 stimulated the recruitment of macrophages and T cells, known facilitators of osteoblast differentiation and survival. Finally, we generated mice harboring a global deletion of Ccl20 and found that Ccl20‐/‐ mice exhibit a reduction in bone mass similar to that observed in Ccr6‐/‐ mice, confirming that this phenomenon is regulated by CCL20 rather than alternate CCR6 ligands. Collectively, these data indicate that CCL20/CCR6 signaling may play an important role in regulating bone mass accrual, potentially by modulating osteoblast maturation, survival, and the recruitment of osteoblast‐supporting cells.

MIP-1δ Activates NFATc1 and Enhances Osteoclastogenesis: Involvement of Both PLCγ2 and NFκB Signaling

Pathological bone resorption is a source of significant morbidity in diseases affecting the skeleton such as rheumatoid arthritis, periodontitis, and cancer metastasis to bone. Evidence indicates that elevated levels of inflammatory mediators such as IL-1, IL-6, and TNF-α play a role in this process by promoting the formation of bone-resorbing osteoclasts. Additionally, current studies have identified inflammatory chemokines of the macrophage inflammatory protein (MIP) family as potential mediators of pathological bone resorption, where both MIP-1α and -3α have been shown to enhance osteoclast (OCL) development. In this study we provide evidence that MIP-1δ, whose expression is associated with renal cell carcinoma bone metastasis and rheumatoid arthritis, enhances OCL formation in vitro via a direct effect on OCL precursors. Consistent with this ability, exposure of OCL precursors to MIP-1δ resulted in the activation of PLCγ2 and NF-κB, two signaling pathways known to regulate OCL differentiation. Moreover, MIP-1δ induced expression and nuclear translocation of NFATc1, a master regulator of osteoclastogenesis, which was dependent on activation of both the PLCγ2 and NFκB signaling pathways. Lastly, consistent with in vitro studies, in vivo administration of MIP-1δ significantly increased OCL number and resorption area as determined using a murine calvarial bone resorption model. Taken together, these data highlight the potential of MIP-1δ as a mediator of pathological bone resorption and provide insight into the molecular mechanism through which MIP-1δ enhances osteoclastogenesis.

Abstract 3394: CITED2: A potential facilitator of breast cancer metastasis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnMetastasis is the ultimate cause of mortality in breast cancer patients. Critical to our ability to prevent and treat metastasis is the identification of the key factors mediating the metastatic process and the understanding of their biological function. Using a murine model of breast cancer metastasis, we recently identified the transcriptional co-activator CITED2 as a potential mediator of bone metastasis. Here, we further explore the potential role of CITED2 in facilitating metastasis using human breast cancer cells. First, we examined the expression of CITED2 mRNA in human breast cancer patient samples by qRT-PCR. Interestingly, CITED2 mRNA levels were significantly (p < 0.05) elevated in primary breast tumors from patients surviving less than 5 years from time of diagnosis (non-survivors, n = 8) relative to those who survived greater than 5 years (survivors, n = 11). Further, mRNA levels of CITED2 were significantly (p < 0.05) elevated in breast cancer metastases (n = 25) relative to both primary breast tumors from survivors and normal mammary organoids. Consistent with mRNA expression, CITED2 protein levels were elevated in primary breast tumors and metastases relative to normal mammary epithelium by immunohistochemical analysis. To investigate the function of CITED2 we generated cell lines in which CITED2 expression was stably increased (CITED2) or decreased (shCITED2) using the human breast cancer cell line MDA-MB-231. While increased expression of CITED2 did not affect cell proliferation in vitro by MTS assay, survival following intracardiac injection was significantly reduced (p < 0.01) suggesting that CITED2 may enhance the ability of cancer cells to survive and grow at secondary sites. Consistent with this hypothesis, we also observed enhanced osteolysis at sites of bone metastasis in animals harboring CITED2 cells relative to vector control. In contrast, stable reduction of CITED2 levels significantly (p < 0.05) inhibited cell proliferation in vitro by MTS assay, consistent with the transcriptional regulation of numerous genes controlling cell cycle events (eg. cyclin E, cdk1, p21, ATM) as observed by qRT-PCR. Moreover, reduced expression of CITED2 resulted in the reversion of MDA-MB-231 cells to a more epithelial-like phenotype as evidenced by a more cuboidal morphology under light microscopy. This observation was further supported by Western analysis demonstrating decreased protein expression of the mesenchymal marker vimentin and increased expression of the epithelial markers E-cadherin and claudin-3. Further, reduction of CITED2 expression inhibited invasive ability by Matrigel invasion assay, while increased levels of CITED2 enhanced invasion. Taken together, these data support a potential role for CITED2 in regulating the metastatic potential of breast cancer cells.nnNote: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3394.