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Effect of IKKα expression on breast cancer patient survival.

cDNA microarray analysis of gene expression between shscramble and shCITED2-expressing MDA-MB-231 cells. Supplementary Fig. S1: Effect of CITED2 knockdown on metastasis to non-bone sites. Supplementary Fig. S2: Regulation of OPN, MMP9, uPA, SPARC, IL-11, and IL-1β by NF-κB signaling in MDA-MB-231 cells. Supplementary Fig. S3: Effect of IKKα expression on breast cancer patient survival.

Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1δ (MIP-1δ), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1δ expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1δ, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1δ stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1δ treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1δ significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1δ expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts. [Cancer Res 2008;68(5):1261–6]

Supplementary Figure 1 Legend from Macrophage Inflammatory Protein–1δ: A Novel Osteoclast Stimulating Factor Secreted by Renal Cell Carcinoma Bone Metastasis

Supplementary Figure 1 from Macrophage Inflammatory Protein–1δ: A Novel Osteoclast Stimulating Factor Secreted by Renal Cell Carcinoma Bone Metastasis

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. © 2016 American Society for Bone and Mineral Research.

The transcriptional co-regulator Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2) may promote breast tumor growth; however, the mechanisms by which its effects are mediated remain to be fully elucidated. Tumor-associated macrophages serve an important function in tumor development and progression and are recruited by chemotactic factors produced by cells within the tumor microenvironment. The present study assessed the effects of CITED2 silencing on macrophage recruitment in two xenograft mouse models of human breast cancer, one in which tumor growth was sensitive to CITED2 silencing (MDA-MB-231) and one in which it was insensitive (MDA-MB-468). The present study identified that silencing CITED2 significantly attenuated macrophage infiltration in MDA-MB-231 but not MDA-MB-468 orthotopic tumors, concordant with its effect on tumor growth. Correspondingly, conditioned media obtained from CITED2-silenced MDA-MB-231 cells exhibited a significantly decreased ability to induce macrophage recruitment by Transwell migration assay, whereas the chemotactic effect of MDA-MB-468 conditioned media was unaffected. Examining the expression of macrophage chemoattractants within orthotopic tumors and tumor cell-conditioned media revealed a significant decrease in C-C motif chemokine ligand (CCL)20 mRNA and protein expression following CITED2-silencing in MDA-MB-231 cells, compared with that in cells transfected with scramble shRNA. However, mRNA and protein expression was unaffected by CITED2-silencing in MDA-MB-468 cells. Furthermore, chromatin immunoprecipitation analysis revealed that CITED2 was localized to the CCL20 promoter in MDA-MB-231 cells, suggesting that it serves a direct function in its regulation, which is consistent with the effect of CITED2 silencing on CCL20 expression. Lastly, neutralizing CCL20 in the conditioned media of MDA-MB-231 cells significantly inhibited macrophage recruitment. Collectively, these results suggest that CITED2 is involved in modulating macrophage recruitment, representing a novel mechanism through which it may influence tumor growth. This may be partly mediated by regulating tumor cell production of the chemokine CCL20.

Raman spectral markers offer new routes to recognition of biomolecular alterations at sites of nascent and progressing metastatic cancer in bone., Breast neoplasms frequently colonize bone and induce development of osteolytic bone lesions by disrupting the homeostasis of the bone microenvironment. This degenerative process can lead to bone pain and pathological bone fracture, a major cause of cancer morbidity and diminished quality of life, which is exacerbated by our limited ability to monitor early metastatic disease in bone and assess fracture risk. Spurred by its label-free, real-time nature and its exquisite molecular specificity, we employed spontaneous Raman spectroscopy to assess and quantify early metastasis driven biochemical alterations to bone composition. As early as two weeks after intracardiac inoculations of MDA-MB-435 breast cancer cells in NOD-SCID mice, Raman spectroscopic measurements in the femur and spine revealed consistent changes in carbonate substitution, overall mineralization as well as crystallinity increase in tumor-bearing bones when compared with their normal counterparts. Our observations reveal the possibility of early stage detection of biochemical changes in the tumor-bearing bones – significantly before morphological variations are captured through radiographic diagnosis. This study paves the way for a better molecular understanding of altered bone remodeling in such metastatic niches, and for further clinical studies with the goal of establishing a non-invasive tool for early metastasis detection and prediction of pathological fracture risk in breast cancer.

Acquired resistance to TGFβ is a key step in the early stages of tumorigenesis. Mutations in TGFβ signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix–loop–helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFβ activity by binding to TGFβ-activated Smads. HeyL−/− mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFβ signaling by Notch acting through HEYL may promote initiation of breast cancer. Cancer Res; 74(22); 6509–18. ©2014 AACR.

CCR6 is important for imiquimod (IMQ)-induced psoriatic dermatitis in mice, which depends on motile CCR6+ γδ T cells that produce IL-17 and IL-22. CCR6 ligands include CCL20 and β-defensins. We used new strains of Ccl20 tdTomato knock-in mice, Ccl20fl/flmice, and Ccr6fl/fl GFP knock-in mice to determine sites of production and action of CCL20 and its role in the localization of γδ T cells and ILCs during imiquimod (IMQ)-induced inflammation. IMQ treatment of Ccl20+/tdT mice led to expression of Ccl20 in keratinocytes (KCs), including in the infundibulum and isthmus of the hair follicles. Ccl20−/− mice showed reduced inflammation and expression of Il17 and Il22 in the treated skin. Deletion of Ccl20 specifically in the KCs by treating Ccl20fl/flK14-CreERT mice with tamoxifen before applying IMQ significantly decreased the accumulation of IL-17-producing gd T cells and ILCs in the epidermis. Depleting KC-derived CCL20 at day 4 when inflammation was well established also reduced the number of epidermal γδ T cells and ILCs. In both cases the percent of IL-17-producing γδ T cells and ILCs did not change, demonstrating that migration to the epidermis and the ability to produce IL-17 are regulated independently. Multi-photon microscopy of IMQ-treated Ccr6+/gfp and Ccr6gfp/gfp(CCR6-deficient) micerevealed a CCR6-independent step whereby dermal CCR6+ cells accumulate at the hair follicles, followed by a CCR6-dependent step whereby the cells move into the superficial epidermis.

// Swaathi Jayaraman 1 , Michele Doucet 1 , Scott L. Kominsky 1 1 Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA Correspondence to: Scott L. Kominsky, email: kominsc@jhmi.edu Keywords: CITED2, primary tumor growth, vasculature, VEGFA isoforms, TGF-β Received: October 13, 2016 Accepted: December 13, 2016 Published: December 20, 2016 ABSTRACT While we previously demonstrated that CITED2 expression in primary breast tumor tissues is elevated relative to normal mammary epithelium and inversely correlated with patient survival, its functional impact on primary tumor development and progression remained unknown. To address this issue, we examined the effect of CITED2 silencing on the growth of human breast cancer cell lines MDA-MB-231 and MDA-MB-468 following orthotopic administration in vivo . Here, we show that CITED2 silencing significantly attenuated MDA-MB-231 primary tumor growth concordant with reduced tumor vascularization, while MDA-MB-468 primary tumor growth and tumor vascularization remained unaffected. Correspondingly, expression of VEGFA was significantly reduced in shCITED2-expressing MDA-MB-231, but not MDA-MB-468 tumors. Consistent with the observed pattern of vascularization and VEGFA expression, we found that TGF-β stimulation induced expression of VEGFA and enhanced CITED2 recruitment to the VEGFA promoter in MDA-MA-231 cells, while failing to induce VEGFA expression in MDA-MB-468 cells. Further supporting its involvement in TGF-β-induced expression of VEGFA, CITED2 silencing prevented TGF-β induction of VEGFA expression in MDA-MB-231 cells. Collectively, these data indicate that CITED2 regulates primary breast tumor growth, likely by influencing tumor vasculature via TGF-β-dependent regulation of VEGFA.

Previously, we identified the transcriptional coactivator CITED2 as a potential facilitator of bone metastasis using a murine mammary cancer model. Extending these studies to human breast cancer, it was observed that CITED2 mRNA expression was significantly elevated in patient specimens of metastatic breast cancer relative to primary tumors, with highest levels in metastasis to bone relative to non-bone sites. To further evaluate CITED2 functions in breast cancer metastasis, CITED2 expression was stably reduced in the human breast cancer cell lines MDA-MB-231 and MDA-MB-468, which are metastatic in animal models. While CITED2 knockdown had no effect on cell proliferation, cell migration and invasion were significantly reduced, as was the establishment of metastasis following intracardiac administration in athymic nude mice. To explore the mechanism behind these effects, gene expression following CITED2 knockdown in MDA-MB-231 cells by cDNA microarray was performed. As confirmed at the mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-κB regulator IKKα was significantly reduced, along with several NF-κB targets with known roles in metastasis (OPN, MMP9, uPA, SPARC, IL11, and IL1β). Furthermore, ChIP assay revealed recruitment of CITED2 to the promoter of IKKα, indicating a direct role in regulating its expression. Consistent with reduced IKKα expression, CITED2 knockdown inhibited both canonical and noncanonical NF-κB signaling. Finally, restoration of IKKα expression following CITED2 knockdown in MDA-MB-231 and MDA-MB-468 cells rescued their invasive ability. Collectively, these data demonstrate that CITED2 modulates metastatic ability in human breast cancer cells, at least in part, through the regulation of IKKα. Implications: The current study highlights the role of CITED2 in facilitating breast cancer metastasis, partly via regulation of IKKα. Mol Cancer Res; 14(8); 730–9. ©2016 AACR.

Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1δ (MIP-1δ), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1δ expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1δ, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1δ stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1δ treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1δ significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1δ expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts. [Cancer Res 2008;68(5):1261–6]

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. © 2016 American Society for Bone and Mineral Research.

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. Introduction: Bone metastasis is a frequent complication and causes marked morbidity in patients with renal cell carcinoma (RCC). Surprisingly, the specific mechanisms of RCC interaction with bone have been scarcely studied despite the inability to prevent or effectively treat bone metastasis. Bone is a reservoir for various growth factors including the pleiotropic cytokine TGF-β1. TGF-β1 has been shown to have tumor-supportive effects on advanced cancers and evidence suggests its involvement in promoting the development of breast cancer bone metastasis. Here, we studied the potential role of TGF-β1 in the growth of RCC bone metastasis (RBM). Materials and Methods: To inhibit TGF-β1 signaling, RBM cells stably expressing a dominant-negative (DN) TGF-βRII cDNA were generated. The in vivo effect of TGF-β1 on RBM tumor growth and osteolysis was determined by histological and radiographic analysis, respectively, of athymic nude mice after intratibial injection of parental, empty vector, or DN RBM cells. The in vitro effect of TGF-β1 on RBM cell growth was determined after TGF-β1 treatment by MTT assay. Results: TGF-β1 and the TGF-β receptors I and II (TGF-βRI/II) were consistently expressed in both RBM tissues and cell lines. Inhibition of TGF-β1 signaling in RBM cells significantly reduced tumor establishment and osteolysis observed in vivo after injection into the murine tibia, although no effect on tumor establishment was observed after injection of RBM cells subcutaneously or into the renal subcapsule. Treatment of five RBM cell lines with TGF-β1 in vitro either had no effect (2/5) or resulted in a significant inhibition (3/5) of cell growth, suggesting that TGF-β1 may promote RBM tumor growth indirectly in vivo. Conclusions: TGF-β1 stimulation of RBM cells plays a role in promoting tumor growth and subsequent osteolysis in vivo, likely through the initiation of tumor-promoting paracrine interactions between tumor cells and the bone microenvironment. These data suggest that inhibition of TGF-β1 signaling may be useful in the treatment of RBM.

B16F10 melanoma is a tumor derived from C57BL/6 mice that has been found to be poorly immunogenic and highly aggressive. Here we have shown that vaccination of mice with irradiated B16F10 cells followed by treatment with a combination of staphylococcal enterotoxins A and B (SEA/SEB) leads to significant and specific protection against subsequent challenge with viable B16F10 cells (at least 25-fold greater than a lethal dose). Also, 75% of mice surviving over 150 days remained tumor-free after rechallenge with viable B16F10 cells, evidence of the development of strong immunologic memory. Additional studies showed increases in CD4+ and CD8+ T-cell populations, cytotoxic T-lymphocyte activity and interferon-γ production, all of which may contribute to enhanced survival. Furthermore, failure to produce protection in either CD4−/− or CD8−/− T-cell knockout mice is evidence that CD4+ and CD8+ T cells play an essential role in induction of immunity. These results show that superantigen administration subsequent to vaccination with inactivated tumor cells results in protective antitumor immunity. Thus, prophylactic vaccination against cancer is a feasible method for arming the immune system prior to the incidence of cancer. © 2001 Wiley-Liss, Inc.

Glioblastoma multiforme is one of the most aggressive and frequently occurring forms of brain cancer. It originates from astrocytes and is characterized by a loss of cell cycle control frequently involving mutations in tumor suppressor genes, such as p53 and p16. Nucleoside analogs, such as acyclovir (ACV), are currently being used in the treatment of viral diseases, such as those caused by members of the herpes family. Further, ACV in combination with type I interferons (IFN) has been shown to be more effective at lower doses in treatment of viral diseases. We show here that ACV at high concentrations (up to 500 microg/ml) inhibited growth in tissue culture of the human glioblastoma cell lines T98G, SNB-19, and U-373 by as much as 68.3% while inhibiting normal human astrocytes by only 38.3%. Related to this, the tumor cells were more than sevenfold more efficient in phosphorylation of ACV to the active phosphate form than normal human astrocytes. Analogous to treatment of virus-infected cells, suboptimal concentrations of ACV were as effective as high concentrations when used in conjunction with low concentrations of IFN-gamma in inhibition of tumor cell growth. At the cellular level, ACV and IFN-gamma inhibited the cell cycle in both the G1 and S phases. The cooperative effect of ACV and IFN-gamma against the glioblastomas appears to be due to direct inhibition of DNA synthesis by ACV in the S phase of the cell cycle and induction by IFN-gamma of the tumor suppressor gene p21wAF1/CIP1, which in turn acts at the level of proliferating cell nuclear antigen (PCNA) and cyclin E/cyclin-dependent kinase 2 (Cdk2) binding and inhibition of function. These studies show that the combination of IFN-gamma and ACV at suboptimal concentrations elicits significant antiproliferative effects on the glioblastoma cell lines T98G, SNB-19, and U-373 while having very little effect on normal human astrocyte cell proliferation.

The antiproliferative effect of IFNα was tested on the human glioblastoma cell lines, U-373MG and T98G. IFNα significantly inhibited the growth of both cell lines, but was more effective in retarding the growth of U-373MG cells. Flow cytometry analysis indicated that synchronized IFNα-treated U-373MG cells showed a strong block in the progression of cells out of the S phase of the cell cycle. T98G cells, on the other hand, showed a moderate delay in the transition of cells from G1 to S phase and only a slight delay in the S phase, consistent with the decreased antiproliferative effect of IFNα on this cell line. IFNα-treated cells were then tested for the induction of the tumor suppressor gene product, p21WAF1/CIP1. Higher levels of p21WAF1/CIP1 were detected in lysates from IFNα-treated U-373MG cells as compared to media controls for as long as 18 h. In IFNα-treated T98G cells, p21WAF1/CIP1 levels were slightly elevated at 4 and 6 h, but decreased to levels similar to controls thereafter, correlating with the antiproliferative effects of IFNα on each cell line. Immunoprecipitation studies on lysates from IFNα-treated U-373MG and T98G cells indicated that increased amounts of p21WAF1/CIP1 were complexed to both cyclin D1 and cyclin E. Further, reduced cyclin-dependent kinase 2 (cdk2) activity was found in both IFNα-treated U-373MG and T98G cells, suggesting a mechanism by which p21WAF1/CIP1 exerted its antiproliferative effects. Lastly, we analyzed the time-dependent production of the cyclins Dl, E, and A. No differences in cyclin D1 levels were found between IFNα-treated and media-treated U-373MG and T98G cells. However, both IFNα-treated U-373MG and T98G cells showed a prolonged elevation in cyclin E, correlating with the G1 to S phase delays observed in these cell lines. Further, the duration of cyclin E production corresponded with the magnitude of the cell cycle delays seen in IFNα-treated U-373MG and T98G cells. Prolonged elevation of cyclin A was also seen in both IFNα-treated U-373MG and T98G cells, the magnitude of which correlated with the S phase delay observed in these cell lines. Thus, the data indicate that IFNα has significant antiproliferative activity against glioblastoma cells that is mediated, at least in part, by the tumor suppressor gene product, p21WAF1/CIP1.

Bone is the most common site of breast cancer metastasis, with bone metastasis occurring in the majority of women with stage IV disease. These patients may experience marked morbidity with pain, decreased mobility, neurologic compromise, and/or pathologic fractures. Although the primary treatment for metastasis is systemic therapy directed at the underlying neoplasm, recent advances in therapy have led to the development of agents tailored to the treatment of bone metastasis including radiopharmaceuticals (eg, strontium-89 chloride) and bisphosphonates (eg, zoledronate or pamidronate). These agents localize predominantly to skeletal areas of high bone turnover like metastatic sites, thereby limiting systemic exposure, but their use is not without toxicity. The administration of bone-targeted radiopharmaceuticals has been associated with painful flare responses and myelosuppres

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.

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.

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. Previously, we identified CITED2 as a novel facilitator of bone metastasis in murine mammary cancer. Extending these initial studies to human breast cancer, CITED2 mRNA expression was significantly elevated in patient samples of metastatic breast cancer relative to primary tumors, with highest levels in bone metastasis. To explore the functional impact of CITED2 in breast cancer metastasis, we stably reduced CITED2 expression in the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 cells, which are metastatic in animal models. While CITED2 knockdown had no effect on cell proliferation, it significantly reduced tumor migration and invasion in vitro and metastasis following intra-cardiac administration in athymic nude mice in vivo. To explore the mechanism responsible for these effects, we examined the impact of CITED2 knockdown on gene expression in MDA-MB-231 cells by microarray analysis. As confirmed at the mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-κB regulator IKKα was significantly reduced along with several downstream targets of the NF-κB pathway (OPN, MMP9, uPA, SPARC, IL11 and IL-1β). In line with the reduced expression of IKKα, both canonical and non-canonical NF-κB signaling was inhibited following CITED2 knockdown. By ChIP assay, CITED2 was recruited to the promoters of IKKα, as well as the NF-κB targets OPN, MMP9, and SPARC. Finally, restoration of IKKα expression in MDA-MB-231 and MDA-MB-468 cells following CITED2 knockdown rescued their invasive ability. Collectively, these results suggest that CITED2 exerts its pro-metastatic effects in breast cancer cells via regulating IKKα. Citation Format: Swaathi Jayaraman, Michele Doucet, Wen M. Lau, Scott L. Kominsky. CITED2 modulates metastatic ability through effects on IKK-alpha in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1623.

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.

Most breast cancers originate in the epithelial cells lining the breast ducts. Intraductal administration of cancer therapeutics would lead to high drug exposure to ductal cells and eliminate preinvasive neoplasms while limiting systemic exposure. We performed preclinical studies in N-methyl-N’-nitrosourea–treated rats to compare the effects of 5-fluorouracil, carboplatin, nanoparticle albumin-bound paclitaxel, and methotrexate to the previously reported efficacy of pegylated liposomal doxorubicin (PLD) on treatment of early and established mammary tumors. Protection from tumor growth was observed with all five agents, with extensive epithelial destruction present only in PLD-treated rats. Concurrently, we initiated a clinical trial to establish the feasibility, safety, and maximum tolerated dose of intraductal PLD. In each eligible woman awaiting mastectomy, we visualized one ductal system and administered dextrose or PLD using a dose-escalation schema (2 to 10 mg). Intraductal administration was successful in 15 of 17 women with no serious adverse events. Our preclinical studies suggest that several agents are candidates for intraductal therapy. Our clinical trial supports the feasibility of intraductal administration of agents in the outpatient setting. If successful, administration of agents directly into the ductal system may allow for “breast-sparing mastectomy” in select women.

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.

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-beta1, a growth factor abundant in the bone microenvironment and known to promote RBM-induced osteolysis in animals. Exposure of RBM1-IT4 cells to TGF-beta1 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-beta type II receptor in RBM1-IT4 cells inhibited MMP-13 expression following TGF-beta1 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-beta1.

Brain metastasis is the most commonly occurring intracranial tumor whose incidence seems to be increasing. With standard therapy, the average survival time of patients is ∼8 months, and treatment often leads to neurologic dysfunction in long-term survivors, emphasizing the need for novel therapeutics. Clostridium perfringens enterotoxin (CPE) has recently been shown to rapidly and specifically destroy cancer cells expressing CPE receptors claudin-3 and claudin-4. Unfortunately, the utility of CPE is precluded by systemic toxicity because its receptors are expressed in numerous organs. Here, we provide the first preclinical evidence that CPE may be uniquely suited to the local treatment of brain metastasis. By immunohistochemical analysis, claudin-3 and claudin-4 were expressed frequently in metastases from breast (15 of 18), lung (15 of 20), and colon (12 of 14) carcinoma, and infrequently in metastases from renal cell carcinoma (2 of 16) and melanoma (2 of 16). In contrast, expression of claudin-3 and claudin-4 was absent in adjacent normal brain tissue. Further examination of the central nervous system (CNS) revealed low or undetectable levels of claudin-3 and claudin-4 in all regions tested by Western and immunohistochemical analysis. Treatment of breast cancer cell lines (MCF-7, MDA-MB-468, NT2.5-luc) and normal human astrocytes with CPE in vitro resulted in rapid and dose-dependent cytolysis exclusively in breast cancer cells, correlating with claudin-3 and claudin-4 expression. Moreover, intracranial CPE treatment significantly inhibited tumor growth and increased survival in two murine models of breast cancer brain metastasis, without any apparent local or systemic toxicity. These data suggest that CPE therapy may have efficacy against a wide variety of brain metastases without CNS toxicity. [Cancer Res 2007;67(17):7977–82]

The development of bone metastasis from renal cell carcinoma (RCC) signals a transition to a terminal state for the patient with previously isolated disease. These patients may suffer the morbidity of severe, persistent pain, pathologic fractures, and spinal compression from vertebral metastasis before they succumb to their cancer. Although recent advancements have been made in the understanding of breast and prostate bone metastasis, there has been less knowledge in the area of metastatic RCC to the skeleton. This particular cancer in bone remains relatively resistant to standard forms of treatment such as radiation and chemotherapy. A better understanding of the biology of RCC bone metastasis is critically needed in order to improve treatment. Bone-derived cell lines and an experimental animal model have been developed in order to explore the relevant mechanisms of how RCC cells survive within and destroy the bone. This review will focus on the growth factor signaling pathways most important for the RCC-stimulated osteoclast-mediated bone destruction, namely the epidermal growth factor receptor (EGF-R) and transforming growth factor-beta receptor (TGF-betaR) pathways. By inhibiting these receptors, growth of RCC within the bone is decreased which, directly or indirectly, decreases bone destruction.

The claudin (CLDN) family of transmembrane proteins plays a critical role in the maintenance of epithelial and endothelial tight junctions. In addition to their function in preserving the structure of tight junctions, CLDNs might also play a role in the maintenance of the cytoskeleton and in cell signalling. Interestingly, several studies have recently reported specific CLDN family members to be overexpressed in a wide variety of cancer types. Although their functional role in cancer progression remains unclear, the differential expression of these proteins between tumour and normal cells, in addition to their membrane localisation, makes them prime candidates for cancer therapy. Preclinical studies have shown that tumour cells overexpressing CLDNs can be successfully targeted via several approaches, including the use of anti-CLDN antibodies as well as the cytolytic enterotoxin from Clostridium perfringens. Further studies are needed to determine the potential systemic toxicity of this approach considering the ubiquitous expression of CLDNs in the body, but CLDN-targeted therapeutics appear to have promise in the treatment of cancer.

In cancer patients and in those at high risk, systemic exposure to agents for therapy or prevention is accompanied by undesirable side effects. We hypothesized that it is possible to prevent and treat breast cancer by introducing anticancer agents into the mammary ductal network. Here, we show the efficacy of intraductally administered anticancer agents 4-hydroxytamoxifen and pegylated liposomal doxorubicin (PLD) in the prevention and treatment of breast cancer using the rat N-methyl-N′-nitrosourea–induced and spontaneous HER-2/neu transgenic mouse (neu-N) models of breast cancer. Intraductal administration of PLD to neu-N mice caused regression of established tumors and prevented tumor development more effectively than i.v. injection (P < 0.0001). Intraductal administration resulted in lower circulating levels of PLD compared with i.v. administration, with no evidence of systemic toxicity or long-term histopathologic changes in the mammary gland. Compared with systemic administration, intraductal injection provides direct access to breast lesions with higher local and lower systemic drug exposure. These studies suggest that this approach has potential for application to prevention and neoadjuvant therapy of early breast cancer. (Cancer Res 2006; 66(2): 638-45)

Purpose: Most often it is not the primary tumor, but metastasis to distant organs that results in the death of breast cancer patients. To characterize molecular alterations in breast cancer metastasis, we investigated the frequency of hypermethylation of five genes (Cyclin D2, RAR-β, Twist, RASSF1A, and HIN-1) in metastasis to four common sites: lymph node, bone, brain, and lung. Experimental Design: Methylation-specific PCR for the five genes was performed on DNA extracted from archival paraffin-embedded specimens of paired primary breast cancer and its lymph nodes (LN) metastasis (n = 25 each); in independent samples of metastasis to the bone (n = 12), brain (n = 8), and lung (n = 10); and in normal bone, brain, and lung (n = 22). Results: No hypermethylation was detected in the five genes in the normal host tissues. In paired samples, LN metastasis had a trend of higher prevalence of methylation compared with the primary breast carcinoma for all five genes with significance for HIN-1 (P = 0.04). Compared with the primary breast carcinomas, all five genes had higher methylation frequencies in the bone, brain, and lung metastasis, with HIN-1 and RAR-β methylation being significantly higher (P < 0.01) in each group. Loss of expression of all five genes correlated, with a few exceptions, to hypermethylation of their promoter sequences in metastatic carcinoma cells microdissected from LNs. Conclusion: The frequent presence of hypermethylated genes in locoregional and distant metastasis could render them particularly susceptible to therapy targeted toward gene reactivation combining demethylating agents, histone deacetylase inhibitors, and/or differentiating agents.

Claudins are transmembrane proteins that seal tight junctions, and are critical for maintaining cell-to-cell adhesion in epithelial cell sheets. However, their role in cancer progression remains largely unexplored. Here, we report that Claudin-7 (CLDN-7) expression is lower in invasive ductal carcinomas (IDC) of the breast than in normal breast epithelium, as determined by both RT-PCR (9/10) and Western analysis (6/8). Immunohistochemical (IHC) analysis of ductal carcinoma in situ (DCIS) and IDC showed that the loss of CLDN-7 expression correlated with histological grade in both DCIS (P

Increasing evidence suggests that superantigens play a role in Immune-mediated diseases. Superantigens are potent activators of CD4* T cells, causing rapid and massive proliferation of cells and cytokine production. This characteristic of superantigens can be exploited in diseases where strong immunologic responses are required, such as in the B16F10 animal model of melanoma. Superantigen administration is able to significantly enhance Ineffective anti-tumor Immune responses, resulting in potent and long-lived protective anti-tumor immunity. However, superantigens are more well-known for the role they play in diseases. Studies using an animal model for neurologic demy-elinatlng diseases such as multiple sclerosis show that superantigens can induce severe relapses and activate auto-reactive T cells not involved in the Initial bout of disease. This may also involve epitope spreading of disease. Superantigens have also been implicated in acute diseases such as food poisoning and TSS, and in chronic diseases such as psoriasis and rheumatoid arthritis. Viral superantigens are also involved in the disease process, including superantigens derived from human Immunodeficiency virus and mouse mammary tumor virus. Finally, immunotherapies that ameliorate the role played by superantigens in disease are discussed.

Glioblastoma is a highly aggressive form of brain cancer characterized by uncontrolled cell growth resulting from a loss of cell cycle regulation. In this study we determined the antiproliferative effects of interferon gamma (IFNgamma) on the glioblastoma cell lines T98G, SNB-19 and U-373, focusing on the ability of IFNgamma to increase levels of p21WAF1/CIP1, an important negative regulator of cell cycle events. IFNgamma was found to inhibit the growth of all cell lines, with inhibition ranging from 82.2% to 45.4%. Flow cytometry analysis showed that IFNgamma treatment caused a cell cycle delay in the G1 or S phases. The strength of this delay varied, correlating with the degree by which IFNgamma inhibited proliferation of each cell line. IFNgamma treatment increased the production of the cyclin dependent kinase inhibitor (CKI) p21WAF1/ CIP1 in all cell lines, the level and kinetics of production of which correlated with the degree and stage of inhibition of cellular proliferation. Further, immunoprecipitation of p21WAF1/CIP1 in complexes of p21WAF1/CIP1/cyclin-dependent kinase 2 (cdk2)/cyclin showed that the amount of p21WAF1/CIP1 in the complexes and the inhibition of cdk2-cyclin kinase activity correlated with the level of p21WAF1/CIP1 produced in the cells by IFNgamma. These results show that IFNgamma has significant antiproliferative effects on the glioblastoma cell lines and suggest that p21WAF1/CIP1 plays a role in mediating these effects.

Acquired resistance to Transforming growth factor-β (TGF-β) is a key step in the early stages of tumorigenesis. Mutations of TGF-β signaling components, often found in other cancers, are rare in breast cancer, and little is known about the development of this resistance in breast cancer. On the other hand, activation of Notch pathway is known to play a substantial role in promoting breast cancer development. We hypothesized that crosstalk between these two pathways occurs through HEYL, a basic helix-loop-helix (bHLH) transcription factor, and a known direct target of Notch. We found that HEYL is overexpressed in 40% of breast cancers. Expression of Notch increased HEYL expression while knockdown of RBP-J, a critical mediator of Notch, reduced HEYL expression in HS578T and MDAMB-231 breast cancer cells. Taking into account the contradictory biological effects of Notch and TGF-β signaling, we sought to examine whether HEYL inhibits the TGF-β pathway. TGF-β treatment or Smad3 overexpression significantly increased the luciferase activity of the TGF-β responsive reporter vector, p3TP-Luc, and the P15 (CDKN2B) gene promoter, but this transactivation was strongly inhibited by HEYL. Bimolecular fluorescence complementation assays confirmed the interaction between Smad 3 and HEYL; immunoprecipitation assays with deletion constructs showed that the Basic domain of HEYL interacts with the MH2 domain of Smad3, and that their interaction is necessary for HEYL to inhibit TGF-β signaling. In addition, using a tet-off inducible breast cancer cell model, HEYL was shown to transcriptionally up-regulate many genes that drive metastasis and tumor angiogenesis. Supporting this concept, HEYL/HER2 double transgenic mice showed nearly 50% increase in lung metastasis (20% vs 46%) compared to HER2/neu mice. Similarly, lung colonization occurred in 100% of mice injected with MDAMB231 while HEYL-shRNAs reduced incidence to 50%. Therefore, HEYL promotes breast cancer growth and metastasis in Smad-dependent and Smad-independent mechanisms. Citation Format: Han Liangfeng, Adam Diehl, Nguyen K. Nguyen, Preethi Korangath, Teo Weiwen, Zhe Zhang, Scott Kominsky, Pedram Argani, Goran Landberg, Saraswati V. Sukumar. The Notch pathway inhibits TGF-β signaling in breast cancer through HEYL-mediated crosstalk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3123. doi:10.1158/1538-7445.AM2013-3123

Metastasis 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. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation 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.