Breast Cancer (BC) initiation, progression and metastasis is accompanied by extensive extracellular matrix (ECM) deposition and remodelling, knows as tumour desmoplasia or fibrosis. Tissue fibrosis is commonly found in aggressive cancers, such as the triple negative breast cancer (TNBC) subtype, and can limit drug delivery to the tumour fuelling treatment resistance, as well as enabling cancer cell invasion and metastasise. In TNBC, the predominant treatment strategy is systemic taxane-based chemotherapy; however, this approach often shows only modest efficacy and can further advance the fibrotic response. The use of anti-fibrotic therapies to target the ECM has therefore gained significant momentum for improving therapy efficacy and extending patient survival.
The Rho-associated protein kinase 1/2 (ROCK 1/2) signalling axis has been shown to contribute to a range of diseases where high tissue fibrosis is a key signature such as pulmonary fibrosis and cardiac hypertrophy. Our laboratory and others have shown that targeting fibrosis and ROCK1/2 signalling in a range of cancer types can impair cancer spread and improve response to standard-of-care chemotherapy; however, poor specificity and drug toxicity of ROCK inhibitors has so far limited successful translation into the clinic. Here, we assess the anti-fibrotic efficacy of a novel, highly specific and clinically relevant ROCK2 inhibitor (ROCK2i).
Using publicly available datasets (TCGA and METABRIC) of BC patient cohorts, we show that high ROCK2 expression significantly correlates with poorer patient survival. Furthermore, single-cell RNA sequencing data from human murine cohorts reveals increased expression of ROCK2 in cancer-associated fibroblasts (CAFs), a key stromal cell type involved in ECM production and remodelling. We also show in organotypic contraction assays that ROCK2i decreases the ability of CAFs to contract a collagen matrix. Here second harmonic generation (SHG) and collagen birefringence imaging reveals that there is a significant decrease in collagen I cross-linking and fibrillar collagen I/III maturation, respectively in ROCK2i treated matrices. Experiments to address whether these ROCK2i-mediated changes in matrix deposition, crosslinking and remodelling will affect subsequent TNBC cell invasion and response to chemotherapy in vitro are ongoing.
In future, this project will interrogate the role that ROCK2 plays in BC progression, invasion and metastasis in vivo. By orthotopically injecting CAFs and cancer cells into the mammary fat pad of immunocompromised mice, we will assess if ROCK2i also reduces matrix crosslinking and remodelling in the TNBC stroma in vivo. This will be combined with innovative intravital imaging techniques, including optical imaging windows, which will be placed above the live mammary tumour or lung to track tumour growth or metastasis, respectively. In order to track TNBC response to chemotherapy we will use a FUCCI cell cycle reporter, which changes colour depending on cell cycle stage. We will then translate our findings into a human disease context where we aim to use patient derived xenografts and cell lines to assess if ROCK2i can be used in a human personalised medicine setting to improve survival in this deadly disease.
Overall, this project will provide exciting pre-clinical data and if successful may provide a new avenue to treat metastatic TNBC.