Robert J. Pauley

Host microenvironment in breast cancer development: Extracellular matrix–stromal cell contribution to neoplastic phenotype of epithelial cells in the breast

Epithelial–mesenchymal interactions play an important role both in normal mammary gland development and during neoplastic transformation. Perturbations in the production, deposition and degradation of the extracellular matrix occurring during neoplastic transformation and progression have been implicated to arise from alterations in the stromal response. These changes in the stroma exhibit a dominant regulatory role, via microenvironmental epigenetic effectors, to contribute to the development of the tumorigenic epithelial phenotype. The role of stromally derived microenvironmental epigenetic effectors in modulating epithelial growth, hormonal response, morphogenesis and epithelial plasticity is discussed.

Aromatase activity and expression in breast cancer and benign breast tissue stromal cells

In situ estrogen synthesis by hormone-dependent breast cancers could potentially regulate cellular proliferation through autocrine or paracrine mechanisms. Several biochemical studies have demonstrated activity of the enzyme aromatase, the rate-limiting step for estrogen synthesis, in breast tumor homogenates. Prior immunohistochemical studies in breast neoplasms demonstrated aromatase antibody binding to both stroma and parenchyma, but biochemically measured enzyme activity significantly correlated only with the level of staining in the stromal component. The present study sought to provide more direct evidence of the predominant role for stromal cell aromatase in breast tumor tissue. Accordingly, breast tumor stromal and epithelial cells were examined for aromatase enzyme activity and messenger ribonucleic acid (mRNA) expression. Stromal and epithelial cells from benign tissue served as a means of comparing activity and regulation in benign and tumor tissue. Enzyme activity in stromal cells from breast tumor tissue was low basally, but increased by 30- to 1200-fold when induced by dexamethasone. Combining dexamethasone with phorbol esters and cAMP produced an additional 1.2- to 4.1-fold stimulation. Analyses of exons III/V and exons IX/X demonstrated that aromatase mRNA expression was also substantially increased by these treatments. Increases in enzyme activity and mRNA expression in cells from benign breast stroma paralleled those observed in tumor stroma, although the increases in enzyme activity were generally lower. In contrast to the responses observed in stromal cells, epithelial cells from breast tumor or nonmalignant breast tissue were unresponsive to dexamethasone, either added alone or in combination with phorbol esters and cAMP. This study provides direct biochemical evidence that aromatase is present in stroma within breast tumors, as in surrounding tissues, and suggests that estrogen synthesis within the tumor may modulate tumor growth via a paracrine mechanism.

Estrogen production via the aromatase enzyme in breast carcinoma: Which cell type is responsible?

Studies of breast tumor homogenates from women with breast cancer have demonstrated the synthesis of estrogens in situ through the enzyme aromatase. The present series of investigations sought to determine which cell type within the tumor is responsible for local estrogen biosynthesis, and whether or not the amount produced is biologically important. Accordingly, we utilized an indirect immunohistochemical scoring method (H-score) to determine the relative amount of enzyme present in tumor epithelial and stromal cells. This revealed a value of 13 for tumor stromal cells and 4.8 for the epithelial component. Contributing to this difference is the fact that a greater percentage of cells in the tumor were stromal (45%) than epithelial (37%). To obtain direct evidence that tumor stromal cells could synthesize estrogens, we isolated and grew these cells in tissue culture. Stromal cells originating from within the tumor could be stimulated by known enhancers of transcription to produce nearly as much aromatase as is found in placental microsomes. Stromal cells isolated from benign tissue distal to the tumor exhibited properties similar to those of the tumor stroma. Epithelial cells, in contrast, did not respond to these enhancers and had low levels of aromatase basally. To obtain proof of the principle that local estrogen synthesis can be biologically meaningful, we measured tumor tissue estradiol levels and growth rates in aromatase-transfected MCF-7 cells implanted into nude mice. Local synthesis resulted in tumor levels ranging from 300 to 800 pg/g and growth rates substantially higher than in non-aromatase-containing tumors. These data suggest that tumor stromal cells contribute the major portion of estrogen synthesized in tumors, and that this local synthesis can increase tumor estradiol levels and growth rates.

Dynamic stromal-epithelial interactions during progression of MCF10DCIS.com xenografts.

MCF10DCIS.com cells form comedo type ductal carcinoma in situ in immune‐deficient mice before forming invasive ductal carcinoma. As the lesions mature, both stromal and epithelial cells undergo phenotypic changes detected by immunohistochemistry. Myofibroblasts are present before the formation of carcinoma in situ and after development of invasive carcinoma. MCF10DCIS. com lesions develop a myoepithelial layer prior to exhibiting a basement membrane surrounding the ductal mass. TGFβ1 is initially expressed by the epithelial cells but is expressed by stroma in invasive carcinoma. Stromal derived factor‐1 is detected in epithelial cells in early carcinoma in situ but is produced in stromal cells in invasive carcinoma. The receptor CXCR4 is expressed by epithelial cells in the xenografts at all times, as is the hepatocyte growth factor receptor c‐met. MCF10DCIS.com xenografts illustrate the dynamic interplay of epithelium and stroma in the development of carcinoma in situ and subsequent invasive carcinoma. Although the phenotype of the epithelial cells may be dependent upon the stroma, the malignant epithelium induces the development of the stroma necessary for progression to the invasive stage.

Rad6B Is a Positive Regulator of β-Catenin Stabilization

Mutations in beta-catenin or other Wnt pathway components that cause beta-catenin accumulation occur rarely in breast cancer. However, there is some evidence of beta-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of beta-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces beta-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, beta-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus-Wnt-1 mammary tumor-derived cells, implicating Rad6B in physiologic regulation of beta-catenin stability and activity. Ubiquitinated beta-catenin was detectable in chromatin immunoprecipitations performed with beta-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of beta-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular beta-catenin but with minimal effects on cell membrane-associated beta-catenin. Measurement of beta-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular beta-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates beta-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of beta-catenin, reveal a positive regulatory feedback loop between Rad6B and beta-catenin and a novel mechanism of beta-catenin stabilization/activation in breast cancer cells.

Expression of transforming growth factor-β receptor type II and tumorigenicity in human breast adenocarcinoma MCF-7 cells

To analyze transforming growth factor‐β (TGF‐β) response during MCF‐7 cell progression, early passage (MCF‐7E, <200 passage) and late passage (MCF‐7L, > 500 passage) cells were compared. MCF‐7E cells showed an IC50 of ∼10 ng/ml of TGF‐β1, whereas MCF‐7L cells were insensitive. MCF‐7E cells contained approximately threefold higher levels of TGF‐β receptor type II (TβRII) mRNA than MCF‐7L, but their TβRI levels were similar. MCF‐7E parental cells showed higher TβRII promoter activity than MCF‐7L cells, which could be attributed to changes in Sp1 nuclear protein levels. Receptor cross‐linking studies indicated that the cell surface receptor levels parallel mRNA levels in both cell lines. Limiting dilution clones of MCF‐7E cells were established to determine the heterogeneity of TβRII expression in this cell line, and they showed varying degrees of TβRII expression. Fibronectin was induced at higher levels in cells expressing higher TβRII levels. All three TGF‐β isoforms were detected in limiting dilution clones and parental cells, but TGF‐β1 was more abundant relative to TGF‐β2 or 3, and no correlation between TGF‐β isoform profile with TGF‐β sensitivity was found. MCF‐7L cells were tumorigenic and formed xenografts rapidly and progressively, whereas MCF‐7E parental and limiting dilution clonal cells showed transient tumor formation followed by regression. These results indicate that decreased TβRII transcription in breast cancer cells leads to a loss of TβRII expression, resulting in cellular resistance to TGF‐β which contributes to escape from negative growth regulation and tumor progression. J. Cell. Physiol. 176:424–434, 1998.

Comedo-ductal carcinoma in situ: A paradoxical role for programmed cell death

Comedo-DCIS is a histologic subtype of preinvasive breast neoplasia that is characterized by prominent apoptotic cell death and has greater malignant potential than other DCIS subtypes. We investigated the mechanisms of apoptosis in comedo-DCIS and its role in conversion of comedo-DCIS to invasive cancer. Clinical comedo-DCIS excisions, and the MCF10DCIS.com human breast cancer model which produces lesions resembling comedo-DCIS were analyzed. Apoptotic luminal and myoepithelial cells were identified by TUNEL and reactivity to cleaved PARP antibody, and cell death assessed by Western blotting, Mitocapture, and immunohistochemical assays. MCF10DCIS.com cells undergo spontaneous apoptosis in vitro, both in monolayers and multicellular spheroids; it is associated with increased mitochondrial membrane permeability, increase in Bax/Bcl-2 ratio, and occurs via caspase-9-dependent P53-independent pathway. This suggests that apoptosis is stromal-independent and that the cells are programmed to undergo apoptosis. Immunostaining with cleaved PARP antibody showed that myoepithelial apoptosis occurs before lesions progress to comedo-DCIS in both clinical comedo-DCIS and in vivo MCF10DCIS.com lesions. Intense staining for MMP-2, MMP-3, MMP-9, and MMP-11 was observed in the stroma and epithelia of solid DCIS lesions prior to conversion to comedo-DCIS in clinical and MCF10DCIS.com lesions. Gelatin zymography showed higher MMP-2 levels in lysates and conditioned media of MCF10DCIS.com cells undergoing apoptosis. These data suggest that signals arising from the outside (microenvironmental) and inside (internal genetic alterations) of the duct act in concert to trigger apoptosis of myoepithelial and luminal epithelial cells. Our findings implicate spontaneous apoptosis in both the etiology and progression of comedo-DCIS. It is possible that spontaneous apoptosis facilitates elimination of cells thus permitting expansion and malignant transformation of cancer cells that are resistant to spontaneous apoptosis.

INT2 and ERBB2 amplification and ERBB2 expression in breast tumors from patients with different outcomes

SummaryThe relationships of INT2 and ERBB2 amplification and of ERBB2 overexpression in primary breast tumors to prognostic factors, recurrence, and survival have generated considerable controversy. The rationale for this study is that long-term, recurrence-free survival is a more direct criterion for testing the validity of a tumor marker than correlation either with prognostic factors or with short-term recurrence and survival. We examined the association of recurrence with INT2 and ERBB2 amplification and ERBB2 expression by comparing primary breast tumors from patients surviving without recurrence for ≥ 8.5 years after diagnosis. the LTS group, to tumors from patients recurring within two years, the RR group. The RR (N = 63) and LTS (N = 61) samples were coded and examined for amplification by Southern blotting and for expression by immunohistochemistry. Comparison between the RR and LTS groups demonstrated that INT2 amplification was associated with a significantly (P = 0.018) higher (5.6-fold) risk of recurrence, an association that remained significant after controlling for lymph node (LN), tumor size (TS), and histograde (HG) status. ERBB2 amplification and expression were not associated with a higher recurrence risk. Survival analyses within the RR group, however, demonstrated significantly shorter survival time among cases with than without ERBB2 amplification (P = 0.018, median survival 16 vs 25 months), or ERBB2 expression (P = 0.019, median survival 15 vs 25 months), but not INT2 amplification. Univariate Cox proportional hazards regression models also demonstrated significantly shorter survival among cases with ERBB2 amplification (P = 0.016) or expression (P = 0.049), that remained significant in multivariate analyses (P = 0.022) for ERBB2 amplification. These results indicate a significant positive association between INT2 amplification and risk for tumor recurrence in the RR as compared to the LTS group. The relationship of ERBB2 amplification or overexpression to patient outcome is more complex. ERBB2 amplification and expression have a significant relationship with shorter survival among patients recurrent within two years, but their occurrence in tumors from women surviving without recurrence for ≥ 8.5 years suggests that ERBB2 status is not predictive of shorter survival for all breast cancers.

Isolation and characterization of a breast progenitor epithelial cell line with robust DNA damage responses

SummaryWe report the establishment of a breast epithelial cell model that undergoes growth arrest at different stages of the cell cycle depending upon the DNA damaging agents encountered. Primary breast epithelial cells from normal reductive mammoplasty were grown in low-calcium culture medium. Free-floating cells under this condition were separated and used for establishment of the MCF-15 breast epithelial cell line. We found that MCF-15 breast epithelial cells display a superb response to different phases of the cell cycle arrest in response to various DNA damaging agents. Immunohistological analysis indicates that MCF-15 cells express cytokeratin 19, CD44, CXCR4, SDF-1, SPARC and vimentin. Although less than 5% of the MCF-15 cells expressed Muc-1 in culture, increased Muc-1 expression was observed in luminal epithelial cells along the newly formed lumen in xenografts. Furthermore, a small population of MCF-15 cells expressed estrogen receptor-α (ERα) in xenografts while ERα expression was not detected in monolayer culture. Therefore, the MCF-15 breast epithelial cell line possesses characteristics of breast progenitor cells and provides a good cell culture model for studying the response to DNA damage and the etiology of aggressive basal-like breast cancers.