Akira Orimo

Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF-1/CXCL12 Secretion

Fibroblasts often constitute the majority of the stromal cells within a breast carcinoma, yet the functional contributions of these cells to tumorigenesis are poorly understood. Using a coimplantation tumor xenograft model, we demonstrate that carcinoma-associated fibroblasts (CAFs) extracted from human breast carcinomas promote the growth of admixed breast carcinoma cells significantly more than do normal mammary fibroblasts derived from the same patients. The CAFs, which exhibit the traits of myofibroblasts, play a central role in promoting the growth of tumor cells through their ability to secrete stromal cell-derived factor 1 (SDF-1); CAFs promote angiogenesis by recruiting endothelial progenitor cells (EPCs) into carcinomas, an effect mediated in part by SDF-1. CAF-secreted SDF-1 also stimulates tumor growth directly, acting through the cognate receptor, CXCR4, which is expressed by carcinoma cells. Our findings indicate that fibroblasts within invasive breast carcinomas contribute to tumor promotion in large part through the secretion of SDF-1.

Stromal Fibroblasts in Cancer: A Novel Tumor-Promoting Cell Type

Tumors are highly complex tissues composed of neoplastic cells and, in the case of carcinomas, stromal cell compartments containing a variety of mesenchymal cells, notably fibroblasts, myofibroblasts, endothelial cells, pericytes, and a variety of inflammatory cells associated with the immune system. Fibroblasts and myofibroblasts often represent the majority of the stromal cells within various types of human carcinomas, yet the specific contributions of these cells to tumor growth are poorly understood. Recent work has demonstrated that stromal fibroblast fractions, named carcinoma-associated fibroblasts (CAFs), that have been extracted from a number of invasive human breast carcinomas are more competent to promote the growth of mammary carcinoma cells and to enhance tumor angiogenesis than are comparable cells derived from outside of these tumor masses. CAFs include large populations of myofibroblasts that secrete elevated levels of stromal cell-derived factor 1 (SDF-1), also called CXCL12, which plays a central role in the promotion of tumor growth and angiogenesis; CAF-derived SDF-1 not only stimulated carcinoma cell growth directly through the CXCR4 receptor displayed on tumor cells but also served to recruit endothelial progenitor cells (EPCs) into tumors, thereby furthering neoangiogenesis. In this review, we highlight the importance of this SDF-1-CXCR4 signaling pathway in the tumor microenvironment and discuss the mechanisms by which stromal fibroblasts within mammary carcinomas enhance tumor growth.

Autocrine TGF-β and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts

Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-β and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-β and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.

Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression

Tumours are highly complex tissues composed of carcinoma cells and surrounding stroma, which is constructed by various different types of mesenchymal cells and an extracellular matrix (ECM). Carcinoma-associated fibroblasts (CAFs), which consist of both fibroblasts and myofibroblasts, are frequently observed in the stroma of human carcinomas, and their presence in large numbers is often associated with the development of high-grade malignancies and poor prognoses. Moreover, in human tumour xenograft models, CAFs extracted from the tumour are more capable of promoting tumour growth through their interactions with carcinoma cells when compared to those isolated from non-cancerous stroma. Taken together, these observations strongly suggest that CAFs actively contribute to tumour progression. In this review we highlight the emerging roles of these cells in promoting tumourigenesis, and we discuss the molecular mechanisms underlying their tumour-promoting capabilities and their cellular origin.

UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.

We have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra‐embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down‐regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post‐coitum in the extra‐embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell‐specific E1A‐like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1‐mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A‐like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF‐2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue‐specific transcriptional coactivator that works in early embryogenesis.

Isolation of estrogen-responsive genes with a CpG island library.

ABSTRACT In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched. CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells. Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively. Both transcripts were increased by estrogen in MCF-7 cells. The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochromec oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5′ upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5′ upstream region of the chicken β-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

Carcinoma-associated fibroblasts: Non-neoplastic tumour-promoting mesenchymal cells†

Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non‐transformed fibroblasts expressing α‐smooth muscle actin (α‐SMA). MFs together with non‐MF cells present in the tumour‐associated stroma are collectively referred to as carcinoma‐associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour‐promoting interactions with other tumour‐constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour‐promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells. J. Cell. Physiol. 228: 1651–1657, 2013.

Molecular Cloning, Structure, and Expression of Mouse Estrogen-responsive Finger Protein Efp CO-LOCALIZATION WITH ESTROGEN RECEPTOR mRNA IN TARGET ORGANS

We have previously identified a human estrogen-responsive gene, efp (estrogen-responsive finger protein), which encodes a putative transcription regulator (Inoue, S., Orimo, A., Hosoi, T., Kondo, S., Toyoshima, H., Kondo, T., Ikegami, A., Ouchi, Y., Orimo, H., and Muramatsu, M.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11117-11121). Here, we report isolation of mouse Efp cDNA and its structure containing three cysteine-rich domains (RING finger and B1 and B2 boxes), a coiled-coil domain, and a C-terminal domain. High levels of Efp mRNA were detected in uterus, ovary, and placenta by RNase protection assay. By in situ hybridization histochemistry the transcripts of efp were also detected in uterus, mammary gland, ovary, and brain, and the co-localization of Efp and estrogen receptor mRNA was particularly demonstrated in these female organs. Moreover, the level of Efp mRNA in uterus and brain, which are known as target organs for estrogen, was up-regulated in vivo by 17β-estradiol. Furthermore, both the Efp and estrogen receptor mRNA were stained in the brain vesicles of 11.5-day embryos by whole mount in situ hybridization. These findings raise the possibility that efp is an estrogen-responsive gene that mediates estrogen action in various target organs.

Carcinoma-Associated Fibroblasts Are a Promising Therapeutic Target

Human carcinomas frequently exhibit significant stromal reactions such as the so-called “desmoplastic stroma” or “reactive stroma”, which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies.

Efp as a primary estrogen-responsive gene in human breast cancer.

We have previously isolated the efp (estrogen‐responsive finger protein) that is required for the normal estrogen‐induced cell proliferation. Here, we show the genomic organization of the human efp gene which consists of nine exons. The efp mRNA was expressed in human breast tumors and the estrogen‐induced expression of the efp was found in MCF‐7 human breast cancer cells. Moreover, efp promoter activity was enhanced through the estrogen‐responsive element dependent on estrogen and estrogen receptor. These results suggest that the efp can mediate estrogen actions such as cell growth in human breast cancer as a primary responsive gene.