Background: Prolactin, but not its best studied mediator STAT5a, is associated with breast cancer progression. Results: In stiff but not compliant collagen matrices, prolactin promotes tumorigenic processes via an enhanced ERK1/2 cascade. Conclusion: Extracellular matrix stiffness powerfully modulates the spectrum of prolactin signals and actions. Significance: Prolactin and stiff matrices interact in a feed-forward loop in breast cancer, suggesting new therapeutic approaches.

BackgroundThe development and progression of estrogen receptor alpha positive (ERα+) breast cancer has been linked epidemiologically to prolactin. However, activation of the canonical mediator of prolactin, STAT5, is associated with more differentiated cancers and better prognoses. We have reported that density/stiffness of the extracellular matrix potently modulates the repertoire of prolactin signals in human ERα + breast cancer cells in vitro: stiff matrices shift the balance from the Janus kinase (JAK)2/STAT5 cascade toward pro-tumor progressive extracellular regulated kinase (ERK)1/2 signals, driving invasion. However, the consequences for behavior of ERα + cancers in vivo are not known.MethodsIn order to investigate the importance of matrix density/stiffness in progression of ERα + cancers, we examined tumor development and progression following orthotopic transplantation of two clonal green fluorescent protein (GFP) + ERα + tumor cell lines derived from prolactin-induced tumors to 8-week-old wild-type FVB/N (WT) or collagen-dense (col1a1 tm1Jae/+) female mice. The latter express a mutant non-cleavable allele of collagen 1a1 “knocked-in” to the col1a1 gene locus, permitting COL1A1 accumulation. We evaluated the effect of the collagen environment on tumor progression by examining circulating tumor cells and lung metastases, activated signaling pathways by immunohistochemistry analysis and immunoblotting, and collagen structure by second harmonic generation microscopy.ResultsERα + primary tumors did not differ in growth rate, histologic type, ERα, or prolactin receptor (PRLR) expression between col1a1 tm1Jae/+ and WT recipients. However, the col1a1 tm1Jae/+ environment significantly increased circulating tumor cells and the number and size of lung metastases at end stage. Tumors in col1a1 tm1Jae/+ recipients displayed reduced STAT5 activation, and higher phosphorylation of ERK1/2 and AKT. Moreover, intratumoral collagen fibers in col1a1 tm1Jae/+ recipients were aligned with tumor projections into the adjacent fat pad, perpendicular to the bulk of the tumor, in contrast to the collagen fibers wrapped around the more uniformly expansive tumors in WT recipients.ConclusionsA collagen-dense extracellular matrix can potently interact with hormonal signals to drive metastasis of ERα + breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0801-1) contains supplementary material, which is available to authorized users.

Biomechanical changes in the tumor microenvironment influence tumor progression and metastases. Collagen content and fiber organization within the tumor stroma are major contributors to biomechanical changes (e., tumor stiffness) and correlated with tumor aggressiveness and outcome. What signals and in what cells control collagen organization within the tumors, and how, is not fully understood. We show in mouse breast tumors that the action of the collagen receptor DDR2 in CAFs controls tumor stiffness by reorganizing collagen fibers specifically at the tumor-stromal boundary. These changes were associated with lung metastases. The action of DDR2 in mouse and human CAFs, and tumors in vivo, was found to influence mechanotransduction by controlling full collagen-binding integrin activation via Rap1-mediated Talin1 and Kindlin2 recruitment. The action of DDR2 in tumor CAFs is thus critical for remodeling collagen fibers at the tumor-stromal boundary to generate a physically permissive tumor microenvironment for tumor cell invasion and metastases.

Breast cancers that express estrogen receptor alpha (ERα+) constitute the majority of breast tumors. Estrogen is a major driver of their growth, and targeting ER-mediated signals is a largely successful primary therapeutic strategy. Nonetheless, ERα+ tumors also result in the most breast cancer mortalities. Other factors, including altered characteristics of the extracellular matrix such as density and orientation and consequences for estrogen crosstalk with other hormones such as prolactin (PRL), may contribute to these poor outcomes. Here we employed defined three dimensional low density/compliant and high density/stiff collagen-I matrices to investigate the effects on 17β-estradiol (E2) activity and PRL/E2 interactions in two well-characterized ERα+/PRLR+ luminal breast cancer cell lines in vitro. We demonstrate that matrix density modulated E2-induced transcripts, but did not alter the growth response. However, matrix density was a potent determinant of the behavioral outcomes of PRL/E2 crosstalk. High density/stiff matrices enhanced PRL/E2-induced growth mediated by increased activation of Src family kinases and insensitivity to the estrogen antagonist, 4-hydroxytamoxifen. It also permitted these hormones in combination to drive invasion and modify the alignment of collagen fibers. In contrast, low density/compliant matrices allowed modest if any cooperation between E2 and PRL to growth and did not permit hormone-induced invasion or collagen reorientation. Our studies demonstrate the power of matrix density to determine the outcomes of hormone actions and suggest that stiff matrices are potent collaborators of estrogen and PRL in progression of ERα+ breast cancer. Our evidence for bidirectional interactions between these hormones and the extracellular matrix provides novel insights into the regulation of the microenvironment of ERα+ breast cancer and suggests new therapeutic approaches.

Estrogen receptor α positive (ERα+) breast cancer accounts for most breast cancer deaths. Both prolactin (PRL) and extracellular matrix (ECM) stiffness/density have been implicated in metastatic progression of this disease. We previously demonstrated that these factors cooperate to fuel processes involved in cancer progression. Culture of ERα+ breast cancer cells in dense/stiff 3D collagen-I matrices shifts the repertoire of PRL signals, and increases crosstalk between PRL and estrogen to promote proliferation and invasion. However, previous work did not distinguish ECM stiffness and collagen density. In order to dissect the ECM features that control PRL signals, we cultured T47D and MCF-7 cells on polyacrylamide hydrogels of varying elastic moduli (stiffness) with varying collagen-I concentrations (ligand density). Increasing stiffness from physiological to pathological significantly augmented PRL-induced phosphorylation of ERK1/2 and the SFK target, FAK-Y925, with only modest effects on pSTAT5. In contrast, higher collagen-I ligand density lowered PRL-induced pSTAT5 with no effect on pERK1/2 or pFAK-Y925. Disrupting focal adhesion signaling decreased PRL signals and PRL/estrogen-induced proliferation more efficiently in stiff, compared to compliant, extracellular environments. These data indicate that matrix stiffness shifts the balance of PRL signals from physiological (JAK2/STAT5) to pathological (FAK/SFK/ERK1/2) by increasing PRL signals through focal adhesions. Together, our studies suggest that PRL signaling to FAK and SFKs may be useful targets in clinical aggressive ERα+ breast carcinomas.