Human Breast Tumor Progression Research Articles

Studying biomineralization pathways in a 3D culture model of breast cancer microcalcifications

Microcalcifications serve as diagnostic markers for breast cancer, yet their formation pathway(s) and role in cancer progression are debated due in part to a lack of relevant 3D culture models that allow studying the extent of cellular regulation over mineralization. Previous studies have suggested processes ranging from dystrophic mineralization associated with cell death to bone-like mineral deposition. Here, we evaluated microcalcification formation in 3D multicellular spheroids, generated from non-malignant, pre-cancer, and invasive cell lines from the MCF10A human breast tumor progression series. The spheroids with greater malignancy potential developed necrotic cores, thus recapitulating spatially distinct viable and non-viable areas known to regulate cellular behavior in tumors in vivo. The spatial distribution of the microcalcifications, as well as their compositions, were characterized using nanoCT, electron-microscopy, and X-ray spectroscopy. Apatite microcalcifications were primarily detected within the viable cell regions and their number and size increased with malignancy potential of the spheroids. Levels of alkaline phosphatase decreased with malignancy potential, whereas levels of osteopontin increased. These findings support a mineralization pathway in which cancer cells induce mineralization in a manner that is linked to their malignancy potential, but that is distinct from physiological osteogenic mineralization.

Abstract PR10: Silencing of semaphorin 7a attenuates breast tumor progression

Abstract Determining what drives conversion of non-lethal to lethal breast cancers is of utmost importance for understanding and blocking tumor metastasis. We have identified semaphorin 7a (Sem7a) as a potent driver of human breast cancer progression, suggesting it may represent a novel target to inhibit metastasis. Sem7a is a GPI membrane anchored protein that promotes attachment and spreading in multiple cell types1,2; previous reports have identified a role for Sem7a in mouse mammary tumorigenesis, including promotion of tumor growth and EMT, as well as induction of angiogenic cytokine production by macrophages and metastatic outgrowth in the lung3-5. However, Sem7a has not been investigated in human breast tumors, so we utilized publicly available microarray datasets and found that SEMA7A gene expression is increased in ~45% of all breast cancers. Further, we found SEMA7A upregulation was associated with poor prognosis, which is consistent with a role in metastasis. Specifically, breast cancer patients with high expression of SEMA7A are at risk for early recurrence and have decreased overall and distant metastasis-free survival rates. We used in vitro and in vivo approaches to assess the role of SEM7a in human breast tumor progression. In vivo, we show that shRNA mediated silencing of Sem7a significantly slows tumor growth, delays tumor progression from in situ to locally invasive disease, and decreases tumor associated lymphangiogenesis. Furthermore, having previously shown that collagen I and COX-2 drive tumor progression in our breast cancer models6,7, we identified a relationship between COX-2 and Sem7a expression at the mRNA and protein levels. Finally, using three- dimensional spheroid and tube formation assays, we show that semaphorin 7a promotes tumor cell invasion on collagen, and lymphangiogenesis, via activation of the β1-integrin receptor. Our results indicate that Sem7a may be novel target for blocking multiple aspects of breast tumor progression that lead to metastasis.

RecQL4 Helicase Amplification Is Involved in Human Breast Tumorigenesis

Breast cancer occur both in hereditary and sporadic forms, and the later one comprises an overwhelming majority of breast cancer cases among women. Numerical and structural alterations involving chromosome 8, with loss of short arm (8p) and gain of long arm (8q), are frequently observed in breast cancer cells and tissues. In this study, we show that most of the human breast tumor cell lines examined display an over representation of 8q24, a chromosomal locus RecQL4 is regionally mapped to, and consequently, a markedly elevated level of RecQL4 expression. An increased RecQL4 mRNA level was also observed in a majority of clinical breast tumor samples (38/43) examined. shRNA-mediated RecQL4 suppression in MDA-MB453 breast cancer cells not only significantly inhibit the in vitro clonogenic survival and in vivo tumorigenicity. Further studies demonstrate that RecQL4 physically interacts with a major survival factor-survivin and its protein level affects survivin expression. Although loss of RecQL4 function due to gene mutations causally linked to occurrence of human RTS with features of premature aging and cancer predisposition, our studies provide the evidence that overexpression of RecQL4 due to gene amplification play a critical role in human breast tumor progression.

Abstract 2812: Macrophage Migration Inhibitory Factor (MIF) regulates monocytic MDSC abundance to contribute to tumor growth and metastasis.

Abstract The inflammatory cytokine, Macrophage Migration Inhibitory Factor (MIF) is a critical mediator of numerous inflammatory disease processes. In addition, MIF is overexpressed in many cancers and the degree of expression correlates with tumor aggressiveness. We identified MIF as a target for inhibition by sulforaphane (SFN), a cancer chemopreventive agent prevalent in broccoli. Based on this observation and the correlation between MIF expression and poor prognosis in human patients, we set out to determine how MIF might contribute to tumor growth and progression. We hypothesized that, as an inflammatory cytokine, MIF may regulate the interaction between the tumor and the host immune response, rather than impacting an intrinsic property of the tumor cells. We are exploring these questions using the syngeneic 4T1 model of breast carcinoma. This model recapitulates many aspects of human breast tumor progression, including spontaneous metastasis from the mammary fat pad to similar target organs. We found that MIF expression in the tumor cells increases tumor growth and metastasis in an orthotopic tumor model. Importantly, the MIF dependent growth differences are only manifested in vivo, as the MIF depleted cell line is not compromised in growth or colony formation in vitro, suggesting that MIF may be contributing to the interaction between the tumor and the host immune response. This was confirmed by demonstrating that MIF depleted and MIF containing tumors exhibit similar growth and metastasis properties in immunodeficient animals. Evaluation of the immune cell infiltrates in the MIF containing and MIF deficient tumors reveal that the abundance of monocytic MDSC within the tumor is dependent on MIF expression. Finally, we demonstrate that depletion of MDSCs abolishes the impact of MIF on tumor growth, suggesting that these cells are critical for MIF to mediate its effects. We are currently exploring the mechanisms by which MIF controls the abundance of monocytic MDSCs in the tumor microenvironment and how this relates to promotion of tumor metastasis. Our work establishes MIF as a potentially valuable therapeutic target in tumor metastasis and supports a potential alternative mechanism for chemoprevention in which SFN may target the inflammatory tumor microenvironment through inhibition of this cytokine. Citation Format: Kendra D. Simpson, Dennis J. Templeton, Janet V. Cross. Macrophage Migration Inhibitory Factor (MIF) regulates monocytic MDSC abundance to contribute to tumor growth and metastasis. [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 2812. doi:10.1158/1538-7445.AM2013-2812

Abstract 4369: The macrophage migration inhibitory factor (MIF) contributes to tumor growth and metastasis though interaction with the host immune response

Abstract The Macrophage Migration Inhibitory Factor (MIF) is overexpressed in many cancers and the degree of expression correlates with tumor aggressiveness. MIF contains a conserved tautomerase activity for which a physiological substrate or function has yet to be identified. We determined that sulforaphane (SFN), a chemopreventive agent prevalent in broccoli, covalently modifies MIF and inhibits its enzymatic tautomerase activity with an IC50 within the concentration range observed in the plasma of human subjects following a broccoli sprout meal. We are examining the role of MIF in tumor growth and metastasis in the 4T1 model of breast carcinoma. This model recapitulates many aspects of human breast tumor progression, including spontaneous metastasis from the mammary fat pad to similar target organs in similar proportions. We found that a 4T1 tumor cell line depleted of MIF forms smaller tumors than the 4T1 cells that express, and is reduced in its ability to metastasize to the lung. Reconstitution of the MIF deficient 4T1 cells with wild-type MIF rescues the defect in tumor growth and metastasis, while a tautomerase-deficient MIF does not. This suggests a function for the tautomerase activity in tumor progression and metastasis. Importantly, the MIF dependent growth differences are only manifested in vivo, as the MIF KD cell line is not compromised in growth or colony formation in vitro, suggesting that MIF may be contributing to the interaction between the tumor and the host immune response. This was confirmed by our demonstration that MIF deficient and MIF containing tumors exhibited similar growth properties in immunodeficient animals. This may represent an alternative mechanism for chemoprevention in which SFN can target the tumor microenvironment rather than the tumor cells. We are currently working to characterize a leukocyte population whose recruitment is altered in MIF depleted tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4369. doi:1538-7445.AM2012-4369

Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-β1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

Epithelial and Stromal Cathepsin K and CXCL14 Expression in Breast Tumor Progression

To evaluate the expression of cathepsin K (CTSK) and CXCL14 in stromal and epithelial cells in human breast tumor progression. We did immunohistochemical analyses of CTSK and CXCL14 expression in normal breast tissue, biopsy sites, benign lesions, ductal carcinoma in situ, and invasive breast tumors of different stages. Expression patterns were related to histopathologic characteristics of the tumors and clinical outcome. The effect of CTSK+ breast stromal fibroblasts on CTSK- breast cancer cells was assessed in coculture. Epithelial expression of CTSK was rarely detected in any of the tissue samples analyzed, whereas CXCL14-positive epithelial cells were found in all tissue types. The expression of CXCL14 was not associated with any tumor or patient characteristics analyzed. Stromal CTSK expression was significantly higher in invasive compared with in situ carcinomas, and in one of the two data sets analyzed, it correlated with higher tumor stage. Among all samples examined, the highest stromal CTSK levels were detected in biopsy sites. Neither epithelial nor stromal expression of CTSK was significantly associated with recurrence-free or overall survival. Coculture of CTSK+ fibroblasts enhanced the invasion of CTSK- breast tumor epithelial cells and this was blocked by CTSK inhibitors. CTSK may function as a paracrine factor in breast tumorigenesis. CTSK+ fibroblasts may play a role in tumor progression by promoting the invasiveness of tumor epithelial cells. The possibility that CTSK inhibitors may have a clinical role in decreasing the risk of tumor progression merits further investigation.

Tamoxifen suppresses histologic progression to atypia and DCIS in MCFIOAT xenografts, a model of early human breast cancer.

We evaluated the effects of tamoxifen on the growth and progression of MCFIOAT xenografts, an estrogen responsive model of human breast tumor progression, in which cells are injected orthotopically into the mammary fat pad of female nude mice. At 10 weeks following implantation, histologic sections of each graft were evaluated microscopically for histologic lesions analogous to human breast tumor progression, graded as simple hyperplasia, complex hyperplasia, atypical hyperplasia, ductal carcinoma in situ and invasive carcinoma. Three out of five xenografts in (endocrine intact) control animals progressed to atypical hyperplasia, one progressed to ductal carcinoma in situ and one to invasive carcinoma. The latter two control grafts also contained foci of putative precursor lesions (i.e. atypical hyperplasia and in situ carcinoma, respectively). Tamoxifen supplemented xenografts (N= 17) were uniformly smaller than controls, but contained invasive carcinoma in a similar proportion (4/17, 24%). However, none of these grafts exhibited ductal carcinoma in situ and only one contained atypical hyperplasia. Most grafts in tamoxifen supplemented animals (10/17, including all four with carcinomas) showed complex hyperplasia, which typically dominated the graft. We conclude that tamoxifen selectively inhibits the appearance or growth of preinvasive index lesions. Development of malignancy in the absence of such precursors, though, implies selection for alternative histogenetic pathways as a result of endocrine manipulation.

Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.

Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer.

Tumor cell-specific loss of p53 protein in a unique in vitro model of human breast tumor progression.

Mutations of the p53 gene are the most frequent genetic lesion in breast cancer. Here, we examined p53 expression in a unique in vitro model of tumor progression derived from a single breast cancer patient (21T series). While the normal mammary epithelial, fibroblast and mesothelial cells derived from this patient expressed easily detectable functional p53 protein, the primary as well as metastatic tumor cell lines demonstrated a lack of p53 protein synthesis. 21T tumor cells failed to exhibit G1 cell cycle arrest upon exposure to gamma-irradiation, and their growth was suppressed by transfection of a normal p53 cDNA, demonstrating a lack of p53-mediated function in these cells. No p53 gene deletion or rearrangements were detectable. PCR and sequence analysis of the entire coding region of p53 gene revealed a novel mutation, an insertion of a single T within codon 33, which resulted in a frame-shift and early termination. The same mutation was observed in all 21T tumor cell lines. These results demonstrate a tumor cell-specific loss of p53 protein due to a frame-shift mutation, and suggest that p53 loss may occur at a relatively early step in breast tumorigenesis before metastatic seeding or emergence of tumor heterogeneity. In addition, the availability of normal and tumor-derived epithelial cells with known p53 sequences from a single breast cancer patient should facilitate understanding of the p53 regulation in mammary cells.