Models Of Inflammatory Breast Cancer Research Articles

Abstract P1-03-09: Combination of nab-Paclitaxel and Bevacizumab Inhibited Tumor Growth and Metastasis of New Inflammatory Breast Cancer Models

Abstract Background: Inflammatory breast cancer (IBC) is a highly lethal form of breast cancer which is characterized by skin redness, irritation, swelling, pain, as well as extensive lymph nodes (LN) and hematogenous metastasis. Animal models of IBC are highly desirable for studying its pathology and for designing effective therapies. In this study, we established two new luciferase-and fluorescently-labeled IBC models and tested the efficacy of the novel drug combination nab-paclitaxel and bevacizumab. Methods: Inflammatory SUM149 breast cancer cells were stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish SUM149-RR, or infected with lentivirus encoding Green Fluorescent Protein (GFP) and Firefly luciferase to establish SUM149- GL. The new cell lines were characterized both in vitro and in vivo. Immunodeficient mice bearing SUM149-RR tumors of 150mm3 in size were treated with saline (control), bevacizumab (4 mg/kg, i.p., twice a week, for 10 weeks), nab-paclitaxel (10 mg/kg, i.v., qdx5), or the combination. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: Luciferase measurements and in vivo imaging showed that both SUM149-RR and-GL clones were highly metastatic to LN, lungs, liver, brain, and spleen. SUM149-RR tumors in control mice displayed ulcerations, edema and redness similar to the clinical disease, while tumors in mice treated with bevacizumab or combination therapy showed no signs of inflammation. Bevacizumab alone decreased tumor growth at later but not early stages of tumor growth, whereas nab-paclitaxel alone inhibited tumor growth by 73%. Combination therapy increased inhibition to 96%, and resulted in 22% (2/9) complete responses. Histologically, tumors from bevacizumab treated groups were more morphologically intact with reduced vascular abnormalities than tumors from control or nab-paclitaxel treated mice. LN and lung metastasis was significantly reduced in all treated groups as compared with control. Conclusions: The SUM149-RR and SUM149-GL lines are new double-tagged models of human IBC that allow organ visualization and accurate quantification of metastasis. These models can be used to study the biology and treatment of IBC. Combination of nab-paclitaxel with bevacizumab was highly effective against SUM149-RR, suggesting the potential usefulness of this regimen for treatment of IBC patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-03-09.

Abstract P6-04-13: Gene and microRNA Signatures of the Mesenchymal Epithelial Transition in Pre-Clinical Models of Inflammatory Breast Cancer

Abstract Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer The pathological hallmark of IBC is lymphovasculogenesis resulting in the invasion of tightly adhered cells, defined as tumor emboli, into lymphovascular spaces of the dermis. IBC tumor emboli have been reported to over-express the glycoprotein E-cadherin, which is at odds with the current hypothesis that tumors with a metastatic phenotype lose E-cadherin, and gain expression of specific transcription factors and microRNAs (miRs) that mediate the process of epithelial mesenchymal transition (EMT). Immunohistochemical staining confirms the presence of E-cadherin on tumor emboli in IBC patient tissues as well as in Mary-X tumor xenografts, which is the only model to recapitulate tumor emboli in vivo. Using unbiased global transcriptional profiling, microRNA microarrays, real time PCR, Western blot validation, immunohistochemistry, immunofluorescence and confocal microscopy, we found that IBC cell lines, and Mary-X tumor emboli from xenografts had significantly elevated expression of genes and their corresponding proteins that collectively mediate tight homotypic cell:cell aggregation, including CDH1 which encodes for E-cadherin, DSC2, which encodes for Desmocollin 2 and JUP/CTNNG1, which encodes for junctional plakoglobin/gamma catenin. Regardless of molecular subtype, IBC cell lines and Mary-X tumor spheroids lacked detectable mRNA or protein for zinc finger E-box binding homeobox 1 gene, ZEB1, with very low levels of ZEB2 gene expression. The expression of CDH1, loss of ZEB1 and low gene expression of ZEB2 by IBC cell lines and Mary-X tumor spheroids was associated with increased expression of specific families of microRNAs including the miR 200 family known to be transcriptional regulators of E-cadherin. In contrast to lack of ZEB1 expression, IBC cell lines expressed either one or all of the SNAI1, SNAI2 and TWIST1 genes, which encode for transcription factors that have been associated with the process of EMT. IBC cell lines that contained ZEB1 cDNA lacked expression of CDH1 and did not produce E-cadherin protein, however the IBC/ZEB1 clones exhibited little difference in their ability to invade across an artificial Matrigel membrane compared to either vector control cells or the parental IBC cells. Taken together, the present observations suggest that pre-clinical models of IBC have distinct gene and microRNA signatures characterized by expression of genes and miRs that collectively support the persistence of compact tightly aggregated tumor cells within IBC tumor emboli, while maintaining an invasive and metastatic phenotype, suggesting that IBC tumor emboli display characteristics of the process of mesenchymal-epithelial transition (MET). Supported in part by the American Airlines-Komen For the Cure Foundation Promise Grant KGO81287 (FMR, MC) and The State of Texas Fund for Rare and Aggressive Breast Cancer (FMR, SK). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-13.

Abstract P1-04-01: X-Linked Inhibitor of Apoptosis Protein (XIAP) Regulates TRAIL-Induced Apoptosis in Inflammatory Breast Cancer Cells

Abstract TRAIL, a proapoptotic receptor agonist, is a promising tool for anticancer therapy due to its selective triggering of death receptor-mediated apoptosis in cancer cells while sparing the majority of normal cells. Overexpression of X-linked inhibitor of apoptosis protein (XIAP) has been identified as a major mechanism of TRAIL resistance as a result of its ability to inhibit caspase activity, the effector arm of apoptotic cell death. Previous studies in our laboratory in preclinical models of inflammatory breast cancer (IBC), a highly invasive and lethal subtype, have revealed that apoptotic dysregulation mediated by XIAP is a key factor in IBC pathogenesis and acquired therapeutic resistance to epidermal growth factor receptor (ErbB1/2) targeting agents. Characterization of TRAIL efficacy in IBC cell lines derived from primary tumors revealed that SUM149, a triple negative, EGFR activated cell line, is sensitive to TRAIL-induced apoptosis and exhibits a corresponding decrease in XIAP and an increase in caspase3 activation compared to TRAIL-resistant SUM190, ErbB2 overexpressing cells in which XIAP levels are unchanged by TRAIL treatment. Profile from an apoptotic proteomic array revealed that SUM190 cells have low basal levels of the TRAIL receptors DR4 and DR5 in comparison to SUM149 cells. Stable overexpression of XIAP in the TRAIL-sensitive SUM149 cells resulted in a two fold decrease in sensitivity to TRAIL with decreased caspase3 activation. Further, SUM149wtXIAP cells showed low levels of TRAIL receptor expression similar to the TRAIL-insensitive SUM190 line. Conversely, increased cell death was observed in SUM190 cells in which XIAP was knocked down using a lentiviral shRNA expression construct. In order to examine therapeutic strategies aimed at restoring function to the apoptotic pathways in IBC cells, we used the small molecule inhibitor Embelin to modulate XIAP action. Treatment of SUM149wtXIAP cells with Embelin in combination with TRAIL enhanced cell death compared to treatment with TRAIL or Embelin alone. Assessment of the interaction using a derivation of the Chou-Talalay method revealed a combination index of 0.054 in SUM149wtXIAP cells, which is indicative of strong synergism between the two agents. These results reveal that knockdown or antagonism of XIAP allows TRAIL to induce activation of effector caspases efficiently and establishes the feasibility of use of XIAP inhibitors to enhance therapeutic apoptosis in IBC therapy. Funded by American Cancer Society-RSG-08-290-01-CCE (GRD), predoctoral DOD BCRP award (KMA), and the Duke University Chancellor's Scholarship (JLA). Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-04-01.

Abstract P5-05-04: E-Cadherin and Its Proteolytic Fragments Trump Epithelial-Mesenchymal Transition and Function as Dominant Oncogenes in Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) and its lymphovascular embolic signature seem to challenge the dogma of prerequisite epithelialmesenchymal transition (EMT) in human breast cancer progression by the apparent maintenance of an epithelial state in all stages of IBC. Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. Although slug, snail and twist are increased in MARY-X spheroids and may decrease E-cadherin transcription to some extent, their effects are neutralized and frankly overridden by the post-transcriptional regulation of E-cadherin which includes its altered degradation and trafficking which, in turn, maintain the epithelial state. Materials and Methods: Because of the uniqueness of the MARY-X model and the observation that E-cadherin was not functioning as a tumor suppressor gene whose loss was triggering EMT but rather whose overexpression was mediating increased survival and resistance to apoptosis, we decided to examine the fate and role of E-cadherin more closely. Results: We discovered that E-cadherin underwent extensive proteolytic processing which resulted in fragments also mediating increased survival and resistance to apoptosis. In both MARY-X and the MARY-X spheroids, Western blot revealed 5 E-cadherin bands: one full length (120 kDa) and four fragments: E-cad/NFT1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa), E-cad/NTF4 (80kDa). Corresponding E-cad/CTF1-4 fragments were also generated. Compared to MARY-X, NFT1 was dramatically increased in the MARY-X spheroids whereas NFT2-4 were significantly decreased. These E-cadherin fragments were produced by specific proteolytic cleavage based on inhibitor studies: NFT1 was produced by calpain, NFT2 by γ-secretase, NFT3 by a matrix metalloproteinase and NFT4 by an unknown mechanism. Predictably levels of calpain activity were significantly increased in the MARY-X spheroids. NFT1 generatedby increased calpain activity retained the p120ctn binding site but lost both the β-catenin and α-binding sites, facilitating its 360° distribution around the cell. Calpain inhibition decreased NFT1 without any affect on full length E-cadherin yet induced disadherence of intact spheroids and prevented the assembly of de novo spheroids. Identical proteolytic fragments of E-cadherin were observed in 3 other human IBC cell lines as well as in lymphovascular emboli of human IBC cases obtained by laser capture microdissection. Discussion: The selective proteolytic processing of E-cadherin ultimately then is responsible for the increased survival of the lymphovascular embolus of IBC and its resistance to chemotherapy-induced apoptosis. Select NT proteolytic fragments may function as adhesion molecules whereas select CT proteolytic fragments may function as transcription factors which confer this survival advantage. E-cadherin and its proteolytic fragments then both trump EMT as well as function as dominant oncogenes in IBC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-04.

Abstract PD02-01: Human Breast Cancer Lymphovascular Tumoral Emboli and Micrometastasis Recapitulate an In Vitro Mammosphere Stem Cell Phenotype

Abstract Background: The existence of stem cells in human cancers has been inferred by clonality experiments and marker studies in vitro and clinical observations in vivo concerning tumor recurrences and emerging drug resistance. We have studied the existence of stem cells in a human model of inflammatory breast cancer, termed MARY-X, a model which forms spheroids which are similar to normal tissue stem cell-derived mammospheres. Comparing MARY-X with common non-IBC breast carcinoma and normal cell lines, we found specific embryonal stem cell markers within the MARY-X spheroids. RT-PCR analyses of MARY-X also revealed the expression of transcriptional determinants essential for the pluripotency and self-renewal of human embryonal stem cells. Since MARY-X spheroids, when injected into mice, form tumors with florid lymphovascular emboli that give rise to micrometastases in numerous organs, we wondered whether tumor emboli and micrometastases from actual human cancers also recapitulated a mammosphere stem cell phenotype. Material and Methods: We carried out laser capture microdissection of tumor emboli and lymph node micrometastases in 200 cases of human breast cancer including cases of both infiltrating ductal as well as lobular cancer. These cases included all of the common molecular classes of breast cancer including luminal A, luminal B, luminal C, Her-2/neu positive and triple negative including the basal subtype. The common denominator which was studied in all of these cases was the lymphovascular embolus and lymph node micrometastasis. Results: By both RT-PCR and IHC, lymphovascular emboli and micrometastases exhibited five-ten fold higher stem cell markers including Stellar, H19, Rex-1, Nestin, CD133 and Aldehyde Dehydrogenase 1 (ALDH1) as well as stem cell transcriptional determinants including OCT4, SOX2, and Nanog than the non-embolic tumoral areas. In addition, stem cell signaling pathways specifically involved in self-renewal and pluripotency including Bmi-1, Hedgehog and Notch 3 were activated selectively within the lymphovascular tumor emboli and micrometastases. These observations held true irrespective of the molecular class of breast cancer from which the embolus was derived and irrespective of the adhesion status (presence or absence of E-cadherin) of the embolus or micrometastasis. Discussion: Our findings indicate that the lymphovascular embolus and micrometastasis is not simply a cellular fragment that detaches from the main tumor but rather represents a selection for a stem cell phenotype. This finding may explain the increased resistance of lymphovascular tumor emboli and micrometastases to chemotherapy and the decreased disease-free survival and poorer prognosis exhibited by patients with significant lymphovascular emboli and micrometastases. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-01.

Angiogenesis, lymphangiogenesis, growth pattern, and tumor emboli in inflammatory breast cancer

This objective of the current review was to provide the reader with a comprehensive summary of the literature related to 3 important and inter-related features of the biology of inflammatory breast cancer (IBC): angiogenesis, lymphangiogenesis, and the formation of tumor emboli. Information derived from animal models of IBC as well as from translational studies using tissue samples of patients with IBC are discussed.

Abstract 2970: Reduced levels of both SNF2L and its isoform SNF2LT mediate the absence of a DNA damage response and resulting apoptosis in the quiescent lymphovascular emboli of inflammatory breast cancer

Abstract Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. In vitro, cells in MARY-X spheroids grow very slowly and can not be cultured as attached monolayers despite the use of numerous cellular and extracellular matrix substrates. Our previous data showed that exogenous growth factors could not stimulate the growth of the spheroids in vitro. The spheroid state itself ultimately may be indirectly responsible for the resistance of the spheroids to chemotherapy since the cells within the spheroids are not actively dividing. However the spheroids also resist apoptosis and there may be other mechanisms in play responsible for their quiescent state. Our laboratory has been studying genes whose expression inversely correlates with E-cadherin expression and observed that both SNF2L and its truncated isoform, SNF2LT, chromatin-remodeling ATPases, are expressed at very low to absent levels in MARY-X and its derived spheroids. Both SNF2L and its truncated isoform, SNF2LT, are nearly ubiquitously expressed in most malignant and normal cell lines, but our previous studies have shown that only malignant cell lines are sensitive to their separate knockdowns. Single knockdowns of either SNF2L or SNF2LT caused DNA damage, a DNA damage response, cell cycle arrest and apoptosis in a number of highly malignant cell lines including Hela, the MDA-MB-468 and −231. Both single and dual knockdown similarly caused DNA damage measured by COMET as well as by increased p-H2AX. However single v dual knockdown had opposite effects on the DNA damage response, cell cycle arrest and apoptosis. Single knockdown of either SNF2L or SNF2LT increased phosphorylated p53 and its downstream genes, GADD45A, 14-3-3σ and p21 whereas double knockdown had no effect. Single knockdown caused a G2/M arrest with upregulation of p-cdc2 whereas double knockdown had no effect. Single knockdown caused profound apoptosis with increased Apaf-1/caspase 9 whereas double knockdown actually increased cellular proliferation. The results suggest that the ratio of SNF2L/SNF2LT determines how a cancer cell responds to DNA damage, either by initiating a damage response which leads to apoptosis or ignoring the DNA damage and continuing to proliferate. Since both SNF2L and SNF2LT are expressed at extremely low levels in MARY-X and its derived spheroids, this mimics the situation of dual knockdown in the other cell lines and hence would be permissive to the occurrence of DNA damage but not a concomitant DNA damage response nor the induction of apoptosis. This quiescent state then contributes to subsequent tumor progression. 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 2970.

Abstract 5091: Lymphovascular tumor emboli exhibit a quiescent state refractory to chemotherapy and targeted therapies because of endogenously low metabolism

Abstract Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. In vitro, MARY-X cells grow very slowly as spheroids and the consumption of nutrients is markedly decreased, suggesting that the metabolism in the MARY-X spheroids is also very low. This low metabolism may also be exhibited in vivo within the lymphovascular embolus and may be responsible for its resistance to chemotherapy. This study investigated the mechanisms which lead to a low level of metabolism by initially analyzing AMPK, a metabolic checkpoint coordinating cell growth with energy status and the mTOR pathway, a pathway that is a highly conserved nutrient-responsive regulator of cell growth found in all eukaryotes. The levels of phosphorylated AMPK proteins (AMPKα and β subunits) decrease gradually with the formation of MARY-X spheroids in vitro. Since it had been reported that E-cadherin inhibits the activity of LKB1, a protein upstream of AMPK, which activates both AMPK and several AMPK-related kinases, the decrease in AMPK activity in the MARY-X spheroids could be the result of the homophilic E-cadherin interactions mediating the tight spheroid aggregates. mTOR activity, on the other hand, increases within the MARY-X spheroids. Because AMPK inversely regulates mTOR activity, it is easy to understand why mTOR activity increases. Using rapamycin on the spheroids led to the down-regulation in the activity of p70 S6 kinase but less effects on the activity of 4E-PB1. Most importantly, the treated spheroids did not dissociate nor undergo apoptosis. Use of Ly294002, a PI3K/Akt inhibitor, completely abolished the activities of both p70 S6 kinase and 4E-BP1, the substrates of mTOR. The treated spheroids dissociated and the cells underwent apoptosis. Compound C (AMPK inhibitor) blocked the activity of AMPK, which resulted in a marked upregulation of the mTOR pathway with great increases in both p70 S6 kinase and 4E-BP1 and an increase in resulting metabolism. However the treated spheroids, just like with Ly294002, dissociated and underwent apoptosis. Increasing the cellular metabolism leads to cell death even in enriched medium. Growing MARY-X in serum free media results in the activation of AMPK, when intracellular ATP levels decline and intracellular AMP increases, as occurs during nutrient deprivation or hypoxia. AMPK activation normally inhibits the mTOR pathway but in MARY-X spheroids grown in serum-free medium, the mTOR pathway is not inhibited, cellular metabolism remains low and the spheroids do not dissociate or undergo apoptosis. If these observations can be confirmed in in vivo studies, we may need to rethink our targeting strategies in the face of a quiescent tumor embolus exhibiting low metabolism. 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 5091.

Abstract 3279: Nab-paclitaxel and bevacizumab treatment in new models of inflammatory breast cancer, SUM149-RR and SUM149-GL

Abstract Background: Inflammatory breast cancer (IBC) is a one of the most lethal forms of breast cancer with a 5-year survival of 40%. The hallmarks of IBC include skin redness, irritation, swelling, pain, as well as extensive lymph nodes (LN) and hematogenous metastasis. Animal models are highly desirable for studying the IBC pathology and for designing therapies that increase patient survival. Here we established and characterized two luciferase- and fluorescently-labeled lines derived from the human IBC line, SUM149. We characterized the growth and metastasis of these lines in vivo as well as their sensitivity to the novel drug combination, nab-paclitaxel and bevacizumab. Methods: SUM149 cells were stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish dual reporter termed RR (RFP and Renilla luciferase), or infected with lentivirus encoding Green Fluorescent Protein (GFP) and Firefly luciferase termed GL (GFP and Firefly Luciferase). The new cell lines (SUM149-RR and SUM149-GL) were characterized in vitro and in vivo after orthotopic implantation into mammary fat pads of immunodeficient mice. SUM149-RR was also examined for sensitivity to nab-paclitaxel alone or in combination with bevacizumab. Mice bearing tumors of 150mm3 in size were treated with saline (control), bevacizumab (4mg/kg i.p., twice a week, for 10 weeks), nab-paclitaxel (10mg/kg, i.v., qdx5), or the combination. Tumor growth was monitored by calipers. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: Luciferase measurements and in vivo imaging showed that both SUM149-RR and -GL clones were highly metastatic to LN, lungs, liver, brain, and spleen. Bevacizumab alone decreased tumor progression at later but not early stages of tumor growth. Nab-paclitaxel alone inhibited tumor growth by 73%. Combination therapy increased inhibition to 96%, and resulted in 22% (2/9) complete responses. SUM149-RR tumors in control mice displayed ulcerations, edema and redness much like the clinical disease. Tumors in mice treated with bevacizumab or combination therapy showed no signs of inflammation. Tumors from bevacizumab treated groups were more morphologically intact with reduced vascular abnormalities than tumors from control or nab-paclitaxel treated mice. LN and lung metastasis was significantly reduced in all treated groups as compared with control. Conclusions: The SUM149-RR and SUM149-GL lines are new double-tagged models for human IBC that allow organ visualization and accurate quantification of metastasis. These models can be used for better understanding of the IBC biology and for developing new therapies tailored to specific pathology of this cancer. Preliminary studies demonstrated high sensitivity of SUM149-RR to nab-paclitaxel/bevacizumab therapy suggesting the potential usefulness for treatment of IBC patients. 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 3279.

Abstract 1776: Lymphovascular tumor emboli exhibit a quiescent state refractory to chemotherapy due to the selective proteolytic processing of E-cadherin

Abstract Previous studies with a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated that overexpressed E-cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super-high density in vitro. In vitro, cells in MARY-X spheroids grow very slowly and can not be cultured as attached monolayers. Our previous data showed that exogenous growth factors could not stimulate the growth of the spheroids in vitro. Whereas treatment with EGF, IGF-1 or TGF-β increases the level of phosphorylated Akt in numerous cell lines which grow as monolayers, treatment of intact MARY-X spheroids with these growth factors actually decreases the levels of phosphorylated Akt. With EGF treatment, while the levels of phosphorylated EGFR increased in the spheroids, indicating that the treatment with EGF results in phosphorylation of its receptor, there is no downstream signaling through Akt. However when the cells within the MARY-X spheroids are dissociated into single cells by a number of different maneuvers, EGF treatment both phosphorylates its receptor and increases the levels of phosphorylated Akt. The results with the other growth factors are similar. It had been reported that E-cadherin-mediated adhesion may inhibit ligand-dependent activation of diverse growth factor receptor tyrosine kinases by decreasing receptor mobility and / or ligand-binding affinity. Because of the uniqueness of the MARY-X model and the role of E-cadherin in possibly regulating this growth factor signaling, we decided to examine E-cadherin more closely. In both MARY-X and the MARY-X spheroids, Western blot revealed 5 E-cadherin bands: one full length (120 kDa) and four fragments: E-cad/NFT1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa), E-cad/NTF4 (80kDa). Compared to MARY-X, NFT1 was dramatically increased in the MARY-X spheroids whereas NFT2-4 were significantly decreased. These E-cadherin fragments were produced by specific proteolytic cleavage based on inhibitor studies: NFT1 was produced by calpain, NFT2 by γ-secretase, NFT3 by a matrix metalloproteinase and NFT4 by an unknown mechanism. Predictably levels of calpain activity were significantly increased in the MARY-X spheroids. NFT1 generated by increased calpain activity retained the p120ctn binding site but lost both the β-catenin and α-binding sites, facilitating its 360° distribution around the cell. Calpain inhibition decreased NFT1 without any affect on full length E-cadherin yet induced disadherence of intact spheroids and prevented the assembly of de novo spheroids. Both of these manipulations converted the cells from being growth factor refractory to growth factor sensitive. The selective proteolytic processing of E-cadherin ultimately then is responsible for the state of quiescence exhibited by the MARY-X spheroids and the associated lymphovascular emboli. 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 1776.

Increased homotypic adhesion and resistance to apoptosis of lymphovascular tumor emboli is due to selective proteolytic processing of E‐cadherin rather than its mere overexpression

Previous studies with a human xenograft model of inflammatory breast cancer, MARY‐X, demonstrated that overexpressed E‐cadherin moderated the formation of the lymphovascular embolus in vivo and tumoral spheroids of super‐high density in vitro. Because of the uniqueness of this model we decided to examine E‐cadherin more closely. In both MARY‐X and the MARY‐X spheroids, Western blot revealed 5 E‐cadherin bands: one full length (120 kDa) and four fragments: E‐cad/NFT1 (100 kDa), E‐cad/NTF2 (95 kDa), E‐cad/NTF3 (85 kDa), E‐cad/NTF4 (80kDa). Compared to MARY‐X, NFT1 was dramatically increased in the MARY‐X spheroids whereas NFT2‐4 were significantly decreased. These E‐cadherin fragments were produced by specific proteolytic cleavage based on inhibitor studies: NFT1 was produced by calpain, NFT2 by γ‐secretase, NFT3 by a matrix metalloproteinase and NFT4 by an unknown mechanism. Predictably levels of calpain activity were significantly increased in the MARY‐X spheroids. NFT1 generated by increased calpain activity retained the p120ctn binding site but lost both the β‐catenin and α‐binding sites, facilitating its 360° distribution around the cell. Calpain inhibition decreased NFT1 without any affect on full length E‐cadherin yet induced disadherence of intact spheroids and prevented the assembly of de novo spheroids. This work was supported by the U.S. Army Breast Cancer Research Program grant W81XWH‐06‐1‐0631.

Evaluation of a CXCR4 antagonist in a xenograft mouse model of inflammatory breast cancer

CXCL12/CXCR4 signaling, being important in the homing of cancer cells to lungs, bone and other organs, is a promising therapeutic target. Our purpose was to determine whether a peptide-based antagonist of CXCR4 would reduce primary tumor growth and/or metastasis in a preclinical mouse model of inflammatory breast cancer. We improved an existing model of inflammatory breast cancer for this study by luciferase transfection of SUM149 cells and the monitoring of such cells in mice by imaging and the luciferase assay. We implanted 2 x 10(6) SUM49-Luc cells along with matrigel into the left thoracic mammary fat pad of nude mice to produce tumors. Our mouse model exhibited important features of inflammatory breast cancer, namely, aggressive local disease, local metastases and distant metastases. To evaluate the efficacy of a CXCR4 antagonist CTCE-9908, by itself or in combination with paclitaxel, we treated groups of ten mice each with CTCE-9908 (25 mg/kg, injected subcutaneously 5 days/week), control peptide SC-9908, paclitaxel (10 mg/kg, injected subcutaneously twice a week), and CTCE-9908 plus paclitaxel concurrently. We assessed all mice weekly by whole-body luciferase imaging to quantify relative primary tumor burden and distant metastases. At the end of the experiment, we quantified primary tumors by weight and lung metastases by luciferase activity assay on tissue lysates. Paclitaxel, a known chemotherapeutic, inhibited primary tumor growth in our model (P < 0.05). CTCE-9908 did not significantly inhibit primary tumor growth or lung metastases as compared to control groups, without or with paclitaxel (P > 0.05). However, CTCE-9908 as a single therapy inhibited organ-specific metastasis to leg (P < 0.05 by chi-squared test and by two-sample t-test).

P53 Mutation in Inflammatory Breast Cancer Cell Lines.

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive manifestation of primary breast cancer and represents 1% to 2% of primary breast cancer in the United States. IBC is characterized by an acute inflammation of the skin of the affected breast generally believed to be caused by blockage of the dermal lymphatics by tumor emboli. Wild type (WT) p53 is a tumor suppressor gene, which induces apoptosis and p53 mutations are associated with poor prognosis in breast cancer. Compared with locally advanced breast cancers, IBC patients have higher levels of mutated p53 protein that has been associated with more aggressive tumors, anthracycline resistance, shorter progression free survival, shorter overall survival, and less favorable long-term outcome. The aim of this study was to determine if there are unique genetic variations in IBC cell lines that would provide specific genetic p53 mutations that could be exploited for targeted therapy with the intent of improving response to treatment and overall survival in IBC.Materials and Methods: Genomic DNA was extracted from six breast cancer cell lines (MDA-453, SUM149, MCF-7, KPL4, MDA321, and SUM190) and the immortalized human mammary epithelial cells (HMLE) using the Qiagen DNA Blood Mini Kit (Valencia, CA). Among the 6 breast cancer cell lines, KPL-4, SUM149 and SUM190 are IBC cell lines; MDA231 and SUM149 have basal-like phenotype; MCF-7 has wild-type p53; SUM190 and KPL-4 are Her2 amplified. The DNA purity and concentration were determined by spectrophotometric measurements of absorbance at 260nm and 280 nm. Polymerase chainreaction (PCR) was performed to amplify the fragments of exons 2-11 of the p53gene using consensus primers. The PCR products were scanned and identified using the Agilent Bioanalyzer 2100. DNA sequencing was performed on PCR products in the ABI PRISM 310 Genetic Analyzer. The BLAST search was used to identify p53 mutations compared with the reference sequence, X54156, from Genbank.Results: We screened 2-11 exon sequences of the p53 gene in the 7 human breast cell lines. We identified two IBC cell lines (SUM149, SUM-190) with a p53 gene alteration that predicted a change in the encoded protein, SUM149 at exon 7 (ATG to ATA, Met-237-Ile) and SUM190 at exon 9 (CAG to TAG, Gln -317-stop). Both mutations have been previously reported. Five nonsense mutations were identified in two other cell lines, MDA-453 and MDA321. No mutations were identified in KPL4 and HMLE cells.Conclusions: The p53 mutation profile in breast cancer cell lines suggests an additional biological feature for the characterization of IBC. Furthermore, these data support the previously reported association between p53 status and chemo- and radioresistance in this disease responsible for poor prognosis. Therapies directed to restore p53 function should be explored in IBC models and in clinical trials. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3161.

Gain in cellular organization of inflammatory breast cancer: A 3D in vitro model that mimics the in vivo metastasis

BackgroundThe initial step of metastasis in carcinomas, often referred to as the epithelial-mesenchymal transition (EMT), occurs via the loss of adherens junctions (e.g. cadherins) by the tumor embolus. This leads to a subsequent loss of cell polarity and cellular differentiation and organization, enabling cells of the embolus to become motile and invasive. However highly malignant inflammatory breast cancer (IBC) over-expresses E-cadherin. The human xenograft model of IBC (MARY-X), like IBC, displays the signature phenotype of an exaggerated degree of lymphovascular invasion (LVI) in situ by tumor emboli. An intact E-cadherin/α, β-catenin axis mediates the tight, compact clump of cells found both in vitro and in vivo as spheroids and tumor emboli, respectively.MethodsUsing electron microscopy and focused ion beam milling to acquire in situ sections, we performed ultrastructural analysis of both an IBC and non-IBC, E-cadherin positive cell line to determine if retention of this adhesion molecule contributed to cellular organization.ResultsHere we report through ultrastructural analysis that IBC exhibits a high degree of cellular organization with polar elements such as apical/lateral positioning of E-cadherin, apical surface microvilli, and tortuous lumen-like (canalis) structures. In contrast, agarose-induced spheroids of MCF-7, a weakly invasive E-cadherin positive breast carcinoma cell line, do not exhibit ultrastructural polar features.ConclusionsThis study has determined that the highly metastatic IBC with an exaggerated malignant phenotype challenges conventional wisdom in that instead of displaying a loss of cellular organization, IBC acquires a highly structured architecture.These findings suggest that the metastatic efficiency might be linked to the formation and maintenance of these architectural features. The comparative architectural features of both the spheroid and embolus of MARY-X provide an in vitro model with tractable in vivo applications.

Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Reverses Mesenchymal to Epithelial Phenotype and Inhibits Metastasis in Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is a rare but aggressive type of advanced breast cancer. Epidermal growth factor receptor (EGFR) expression is an independent poor prognostic factor in IBC. The purpose of this study was to determine the effect on IBC tumorigenicity and metastasis of blocking the EGFR pathway. IBC cell lines, which express high level of EGFR, were treated with EGFR small interfering RNA and with the EGFR tyrosine kinase inhibitor erlotinib. The role of EGFR in IBC cell proliferation, motility, invasiveness, and change of the expression levels of epithelial-mesenchymal transition markers was examined. The role of extracellular signal-regulated kinase (ERK)-1/2 in erlotinib activity was also studied. The activity of erlotinib in tumor growth and metastasis was examined in an orthotopic xenograft model of IBC. Erlotinib inhibited proliferation and anchorage-independent growth of IBC cells, and this inhibition was ERK dependent. Erlotinib inhibited cell motility and invasiveness and reversed the mesenchymal phenotype of IBC cells to epithelial phenotype in three-dimensional culture. Erlotinib dramatically inhibited IBC tumor growth in a xenograft model. Interestingly, erlotinib inhibited spontaneous lung metastasis, even at a low dose that had no significant effect on primary tumor growth. These erlotinib-treated tumors were converted to epithelial phenotype from mesenchymal phenotype. The EGFR pathway is involved in tumor growth and metastasis of IBC. Targeting EGFR through the ERK pathway may represent an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in EGFR-expressing IBC.

Inflammatory breast carcinoma as a model of accelerated self-metastatic expansion by intravascular growth

Inflammatory breast carcinoma as a model of accelerated self-metastatic expansion by intravascular growth

Suppression of mesenchymal phenotype in inflammatory breast cancer by EGFR tyrosine kinase inhibitor erlotinib: a novel approach to suppressing metastasis.

Abstract Abstract #2042 Background: Inflammatory breast cancer (IBC) is a rare subtype well known for its propensity for rapid metastasis. The cause of this rapid metastasis in IBC is unknown. An immunohistochemical analysis of 44 cases of IBC showed HER2 overexpression in 48% of patients and epidermal growth factor receptor (EGFR) overexpression in 30% of patients. EGFR overexpression was the only poor prognostic factor: the 5-year overall survival rate was significantly lower for women with EGFR-positive disease than for women with EGFR-negative disease (P=0.01). HER2 overexpression was not a prognostic factor. The association between EGFR overexpression and increased risk of death indicated that EGFR may represent a potential therapeutic target in IBC. We hypothesized that suppression of the EGFR pathway inhibits proliferation and metastasis of IBC.&amp;#x2028; Methods: SUM149 IBC cells, which express low levels of HER2 and high levels of EGFR, were treated with siRNA against EGFR and with the EGFR tyrosine kinase inhibitor erlotinib.&amp;#x2028; Results: EGFR siRNA knockdown (but not non-targeting siRNA control) inhibited the proliferation of SUM149 cells. SUM149 cells were sensitive to EGFR tyrosine kinase inhibitor erlotinib in a 2-dimensional (2-D) culture system (median inhibitory concentration [IC50] = 0.90 µM). When we activated ERK by transfecting constitutively active MEK1 in SUM149 cells, the cells showed more resistance to erlotinib. Moreover, ERK siRNA knockdown sensitized SUM149 cells to erlotinib. Further, when we cultured the SUM149 cells in matrigel by using the 3-D culture system (100% matrigel in the bottom layer and cultured medium with 5% FBS and 2% matrigel in the top layer), erlotinib treatment changed the molecular phenotype of SUM149 cells from mesenchymal (a phenotype characterized by low beta-catenin expression and high vimentin and fibronectin expression) to epithelial (recovery of beta-catenin to the sites of cell-cell contacts; downregulation of fibronectin). This reversal of the mesenchymal phenotype, a hallmark of inhibition of epithelial-to-mesenchymal transition (EMT), was ERK dependent. Interestingly, the erlotinib concentration that inhibited the mesenchymal phenotype (0.1 mcM) was one log lower than the concentration that inhibited proliferation (1 mcM).&amp;#x2028; Conclusion: Inhibition of tumor growth and EMT in SUM149 IBC cell lines is dependent on the ERK pathway through the EGFR pathway. The erlotinib dose needed to produce an anti-mesenchymal effect is much lower than the cytotoxic dose. Thus, we speculate ERK pathway to be important in inhibiting metastasis in IBC. We are currently investigating the effects of erlotinib in a xenograft model of IBC. Our study provides a rationale for developing novel treatment strategies targeting the EGFR and ERK pathways to inhibit the growth and metastasis of IBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2042.

Validation of a hierarchical stem cell population in inflammatory breast cancer (IBC) by comparative genomic analysis

The existence of stem cells in human solid tumors has been presumed based on clinical observations and biomarker and clonal dilution studies. Our previous studies with a human xenograft model (MARY‐X) of IBC, a model that forms spheroids in vitro that resemble a blastocyst, have demonstrated the presence of a prominent stem cell‐like population of cells. Comparing MARY‐X with non‐IBC breast carcinoma and normal lines, we found known embryonal stem cell markers (Stellar, H19, Rex‐1, Nestin, CD133) present. RT‐PCR analysis also revealed the expression of OCT4, SOX2, and Nanog, transcriptional determinants essential for the pluripotency and self‐renewal of human embryonal stem cells. However these findings do not prove that there is truly a hierarchical reserve stem cell population since both stem cell markers and functions could be reversibly derived from the proliferating population. We reasoned that true hierarchical stem cells should have fewer genomic alterations (gains and losses) than the proliferating population. To this end we conducted array CGH and identified 10 regions of amplification and 10 regions of loss. We then derived probes from each of these regions and conducted multicolor FISH on individual cells disadhered from the spheroids. Each of the probes recognized a specific chromosomal region in normal HMECs. All of the probes derived from amplified regions or deleted regions showed marked amplification (5–20 fold) or loss in &gt;99% of the cells. However there was a distinct subpopulation of &lt;1% of human cancer cells that contained markedly fewer genomic alterations (gains or losses). The existence of this distinct subpopulation suggests that a truly hierarchical stem cell population exists in IBC. This comparative genomic approach may be generally applicable to the identification and study of stem cells in many solid tumors.

Reversible Model of Spheroid Formation Allows for High Efficiency of Gene DeliveryEx Vivoand Accurate Gene AssessmentIn Vivo

The native three-dimensional architecture of carcinomas, which governs numerous autocrine-paracrine interactions related to tumor progression, cannot be faithfully recreated in most in vitro models. Even when the three-dimensional architecture is recreated in artificial scaffolds such as soft agar, this approach does not truly recreate the natural microenvironment of the tumor. Multicellular spheroids can reasonably recreate in vitro the natural three-dimensional architecture of carcinomas, but even the most efficient gene delivery vectors will penetrate only the outer layers of these structures and hence only a small fraction of cells will receive the gene of interest. If the multicellular spheroids are disrupted into a single-cell suspension in order to achieve high transfection efficiency, the single-cell production may have so altered the gene expression profile of the spheroid as to bring into question its present relevancy to in vivo tumor progression. Our laboratory has developed a human-SCID (severe combined immunodeficient) mouse model of inflammatory breast cancer, MARY-X, which grows as tight multicellular spheroids in vitro and as lymphovascular emboli in vivo. The spheroids, which express only low levels of surface sialyl-Lewis(x/a) (sLe(x/a)), are able to form compact homotypic cell clumps mediated by an intact, overexpressed E-cadherin/alpha,beta-catenin axis. The spheroids can be fully disrupted by trypsin proteolysis, anti-E-cadherin antibodies, or Ca(2+) depletion. Of these approaches the disruption with depleted Ca(2+), complete after 30 min, is fully reversible by the readdition of Ca(2+) within 6 hr. This time interval allows for a transfection "window" in which successful gene delivery can be achieved before spheroid reformation. Retroviruses (10(6)-10(7) CFU/ml) carrying the gene encoding either green fluorescent protein (GFP), a dominant-negative E-cadherin mutant (H-2K(d)-E-cad), its control (H-2K(d)-E-cad Delta C25), or alpha-1,3-fucosyltransferase III (FucT-III), an enzyme that increases surface sLe(x/a), were used to transfect either intact (wild-type) or disadhered/readhered (reformed) spheroids. There were marked differences in transfection efficiency in the reformed versus wild-type spheroids. Retroviral transfection of GFP resulted in successful delivery of this reporter gene to only the outer layer of cells of the wild-type spheroids, but to all layers of the reformed spheroids. A single retroviral transfection of H-2K(d)-E-cad, H-2K(d)-E-cad Delta C25, or FucT-III produced evidence of their respective gene expression at 72 hr throughout all layers of the reformed spheroids, but only H-2K(d)-E-cad and FucT-III produced progressive disadherence. Both H-2K(d)-E-cad-MARY-X and FucT-III-MARY-X lost their ability to form lymphovascular emboli in SCID mice. This reversible model of spheroid formation has provided us with insight into the pathogenesis of inflammatory breast carcinoma. If more broadly applied, this model could be used to examine the effects of any gene, using any gene delivery system in the three-dimensional context of native tumoral architecture.