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Modeling breast cancer in general and hormone-sensitive breast cancer, in particular in mice, has several limitations. These are related to the inbred nature of laboratory mice, and do not allow adequate appreciation of the contribution of the host's genetic heterogeneity in tumor growth. In addition, the naturally low estrogen levels of mice makes estradiol supplementation obligatory for tumor growth. Here, we show that Peromyscus californicus , following cyclosporine-mediated immunosuppression, supports the growth of both MDA-MB-231 estrogen-independent and MCF7 estrogen receptor-positive breast cancers without exogenous estradiol supplementation. Tumor growth was inhibited by fulvestrant or letrozole, confirming that MCF7 xenografts remain hormone dependent in vivo and suggesting that P. californicus can be used as an alternative to conventional mice for the study of hormone-sensitive breast cancer. The fact that Peromyscus stocks are outbred also facilitates the study of breast cancer in genetically heterogenous populations., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.

The migration of cancer cells towards gradients of chemoattractive factors represents a potential, yet elusive, mechanism that may contribute to cancer cell dissemination. Here we provide evidence for the maintenance of a gradient of increasing CCL8 concentration between the epithelium, the stroma and the periphery that is instrumental for breast cancer cells' dissemination. In response to signals elicited by the neoplastic epithelium, CCL8 production is enhanced in stromal fibroblasts at the tumor margins and in tissues at which breast cancer cells tend to metastasize such as the lungs and the brain. Manipulation of CCL8 activity influences the histology of the tumors and promotes major steps of the metastatic process such as invasion to adjacent stroma, intravasation and ultimately extravasation and seeding. These findings exemplify how gradients of chemoattractive factors such as CCL8, drive metastasis and suggest that interference with their operation may provide means for breast cancer management., Competing Interests: The authors declare no conflict of interest.

The synthesis of M(I)(CO)(3)(NNO) (M = Re, (99m)Tc) complexes conjugated to the antitumor agent 2-(4'-aminophenyl)benzothiazole and to its 6-methyl derivative, as well as their in vitro and in vivo biological evaluation as breast cancer radiopharmaceuticals, is reported. The Re complexes displayed under the fluorescence microscope clear uptake by the sensitive to the 2-(4'-aminophenyl)benzothiazole pharmacophore breast cancer cell lines MCF-7 and T47D, while uptake by less sensitive lines and by normal fibroblasts was much weaker. In accordance, uptake of the corresponding radioactive (99m)Tc complexes was clearly higher in the breast cancer cell lines MCF-7 and MDA-231 compared to normal fibroblasts. Biodistribution of the (99m)Tc complexes in SCID mice bearing MCF-7 xenografts showed appreciable tumor uptake. A tumor/muscle ratio of 2.2 was measured for the complex conjugated to 2-(4'-aminophenyl)benzothiazole that led to successful tumor imaging. The results render the 2-(4'-aminophenyl)benzothiazole complexes potential candidates for imaging ((99m)Tc) and targeted radiotherapy ((188)Re) of breast cancer.

During carcinogenesis, stromal fibroblasts undergo certain changes in concert with their neoplastic neighbors, an interaction that progressively leads to a cancer-associated state. However, despite the increasing appreciation of the importance of stromal/tumor interactions in the progression of cancer, little is known about the factors responsible for regulating the crosstalk between stromal fibroblasts and neoplastic cells. Here we show that the stage of the disease in primary human breast lesions affects p21 expression in the fibroblasts. In stromal fibroblasts of benign fibroadenomas, p21 exhibits a periductal pattern of staining, which is abolished in malignant adenocarcinomas in which p21 immunopositivity exhibits a mosaic pattern that eventually is abolished in more aggressive types of the disease. In order to address the role of fibroblasts' p21 in tumorigenesis, we have reconstituted MCF7 human breast cancers in mice, with fibroblasts differing in the p21 status. These experiments showed that p21 deficiency in stromal fibroblasts accelerates tumor growth through cell non-autonomous mechanism(s). In addition, even a transient, siRNA-mediated p21 suppression in fibroblasts sufficiently stimulates MCF7 and MDA-MB-231 growth in vivo. We propose that p21 regulation is intimately linked with the ability of stromal cells to affect tumor growth.

Cancer cells are committed to an actively secretory state that facilitates communication with their microenvironment. We have addressed the role of ERp29, a novel endoplasmic reticulum secretion factor in mammary carcinogenesis using MCF-7 human breast cancer cells as a model. Xenografts originating from cells stably transfected with dominant-negative form of ERp29 were smaller and better differentiated than those derived from cells overexpressing wild-type ERp29. Similar effects were observed by siRNA-mediated ERp29 silencing in xenografts. However, unlike xenografts, the modulation of ERp29 in vitro did not affect the rate of cell proliferation. In addition, we have evaluated the expression of ERp29 in the resting and lactating mammary glands of mice as well as in the human primary breast tumors. About 25% of breast cancers and also lactating mammary glands were expressing ERp29 while the resting glands did not. Taken together these data suggest the active involvement of ERp29 in the malignant conversion of mammary epithelial cells.

The efficacy of chemotherapy is usually viewed as the outcome of cancer-cell-autonomous processes while the contribution of stroma is being overseen. Here we show that p53 mutations in stromal fibroblasts, a genetic lesion that is detectable in primary breast, prostate and probably other cancers, while they accelerate tumorigenesis they also sensitize tumours against conventional chemotherapy by doxorubicin and cis-platinum. The mechanism by which p53 of stromal fibroblasts affects the response of a tumour against chemotherapy is likely to involve the induction of senescence in the fibroblasts which in turns results in the production of growth factors acting onto the cancer cells by paracrine mechanisms. Our findings identify stromal fibroblasts as important modulators of the efficacy of anticancer therapy., ((c) 2008 Wiley-Liss, Inc.)

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as an autocrine/paracrine growth factor for various human cancers. A splice variant (SV) of the full-length receptor for GHRH (GHRHR) is widely expressed in various primary human cancers and established cancer cell lines and appears to mediate the proliferative effects of GHRH. To investigate in greater detail the role of SV1 in tumorigenesis, we have expressed the full-length GHRHR and its SV1 in MCF-7 human breast cancer cells that do not possess either GHRHR or SV1. In accordance with previous findings, the expression of both GHRHR and SV1 restored the sensitivity to GHRH-induced stimulation of cell proliferation, with SV1 being more potent than the GHRHR. Furthermore, MCF-7 cells transfected with SV1 proliferated more quickly than the controls, even in the absence of exogenously added GHRH, suggesting the existence of intrinsic, ligand-independent activity of SV1 after its transfection. In agreement with the stimulation of cell proliferation, the levels of proliferation markers cyclin D1, cyclin E, and proliferating cell nuclear antigen were elevated in MCF-7 cells treated with GHRH, cultured in both serum-free and serum-containing media. In addition, SV1 caused a considerable stimulation of the ability of MCF-7 cells to grow in semisolid medium, an assay considered diagnostic for cell transformation. Collectively, our findings show that the expression of SV1 confers oncogenic activity and provide further evidence that GHRH operates as a growth factor in breast cancer and probably other cancers as well.

Prostate, breast, and probably other epithelial tumors harbor inactivating mutations in the p53 tumor suppressor gene in the stromal cells, implying the nonautonomous action of p53 in carcinogenesis. We have tested this hypothesis by evaluating the tumorigenicity of MCF7 human breast cancer cells in severe combined immunodeficient mice that differ in their p53 status. Our results showed that, indeed, p53 ablation in the hosts reduced the latency for the development of MCF7 tumors. Furthermore, we show that heterozygous hosts frequently undergo loss of heterozygosity at the p53 locus in the tumor stroma tissue by mechanism that resembles the inactivation of p53 in primary tumors. To evaluate the impact of p53 ablation in the stromal fibroblasts, in tumorigenesis, tumors were reconstituted in mice bearing wild-type p53 alleles, by mixing MCF7 cells with fibroblasts isolated from mutant or wild-type p53 mice. Our results suggest that tumors containing p53-deficient fibroblasts developed faster and were more aggressive than their counterparts with wild-type fibroblasts, although their neoplastic component, namely MCF7 mammary carcinoma cells, was identical in both cases. These data strongly support the notion for the operation of a nonautonomous mechanism for p53 action in primary tumors and provide a mechanistic association between p53 mutations in the stromal component of epithelial tumors and carcinogenesis.

Objective: GHRH is secreted by the hypothalamus and, upon binding to specific GHRH receptors in the pituitary, stimulates growth hormone (GH) production and release from the pituitary. In addition to this neuroendocrine action, accumulated evidence implies additional roles for GHRH in carcinogenesis in non-pituitary tissues. In vitro and in vivo studies have shown that splice variant 1 (SV1) of the GHRH receptor, which is widely expressed in non-pituitary tissues and cancers, can mediate the proliferative effects of GHRH. The aim of the present study was to investigate the operation of an autocrine stimulatory loop between GHRH and SV1 in primary breast tumors., Design: Fifty-three primary breast tumors were evaluated for GHRH and SV1 expression., Methods: Expression of GHRH and SV1 was assessed by immunohistochemistry using anti-GHRH SV95 and anti-SV1 2317/5 polyclonal antibodies., Results: About 40% of the specimens tested express GHRH and/or SV1 (approx. 25% each), while in 35% of these positive specimens co-expression of these antigens was detected (P<0.01). Furthermore, a correlation of GHRH, but not SV1, expression was detected in lobular compared with ductal carcinomas., Conclusions: These results constitute the first demonstration for the expression of GHRH and SV1 in primary breast cancers, and provide evidence for the operation of an autocrine stimulatory loop between GHRH and SV1 in primary cancers. Our findings indicate that GHRH analogs could have diagnostic and therapeutic applications for the management of breast cancer.

Allelic imbalance or loss of heterozygosity (LOH) studies have been used extensively to identify regions on chromosomes that may contain putative tumour suppressor genes. We looked for evidence of microsatellite instability (MI) and LOH on chromosome 7q, 10q, 11p and 17q using seven polymorphic microsatellite markers. In 42 paired breast cancer-peripheral blood DNA samples we identified 24 tumours (57%) exhibiting genetic alterations. Twenty-one specimens exhibited LOH (50%), while 11 specimens exhibited MI (26%) in at least one microsatellite marker. The most frequent incidence of LOH was found for the marker THRA1 (8/33, 24%) indicating that thra I gene becomes a strong candidate tumour suppressor gene, whereas of MI it was D10S109 (3/26, 12%). These MI and LOH data were analysed using a range of clinicopathological parameters. Tumours displaying MI with no evidence of LOH and tumours exhibiting MI and LOH belonging to stage II or III were found, however none were at stage I. These data suggest that MI may be an early event in mammary tumorigenesis whereas LOH occurs at a late stage. A significant association between the absence of oestrogen receptors (p < 0.01) and the absence of both oestrogen and progesterone receptors (p < 0.001) at 17q21 were observed, indicating a possible relationship between specific genetic changes at this region and hormonal deregulation in the progression of breast cancer.