The role of PKC and PKD in CXCL12 and CXCL13 directed malignant melanoma and acute monocytic leukemic cancer cell migration.

Cancer metastasis is the leading cause of cancer related mortality. Chemokine receptors and proteins in their downstream signalling axis represent desirable therapeutic targets for the prevention of metastasis. Despite this, current therapeutics have experienced limited success in clinical trials due to a lack of insight into the downstream signalling pathway of specific chemokine receptor cascades in different tumours. In this study, we investigated the role of protein kinase C (PKC) and protein kinase D (PKD) in CXCL12 and CXCL13 stimulated SK-MEL-28 (malignant melanoma) and THP-1 (acute monocytic leukaemia) cell migration. While PKC and PKD had no active role in CXCL12 or CXCL13 stimulated THP-1 cell migration, PKC and PKD inhibition reduced CXCL12 stimulated migration and caused profound effects upon the cytoskeleton of SK-MEL-28 cells. Furthermore, only PKC and not PKD inhibition reduced CXCL13 stimulated migration in SK-MEL-28 cells however PKC inhibition failed to stimulate any changes to the actin cytoskeleton. These findings indicate that PKC inhibitors would be a useful therapeutic for the prevention of both CXCL12 and CXCL13 stimulated migration and PKD inhibitors for CXCL12 stimulated migration in malignant melanoma.

The development of potent, competitive CXCR4 antagonists for the prevention of cancer metastasis.

Cancer metastasis is the cause of up to 90 % of cancer related mortality. The CXCR4 receptor and its cognate ligand, CXCL12, have major roles in enabling cancer metastasis and consequently, the CXCR4 receptor has become an attractive therapeutic target for the prevention of metastasis. Despite this, CXCR4 antagonists have had limited success in clinical trials due to cellular toxicity and poor stability and efficacy. In this study, we developed a novel, competitive CXCR4 antagonist (IS4) that through copper-catalysed-azide-alkyne-cycloaddition can be clicked to other chemical moieties such as fluorescent dyes (IS4-FAM) for CXCR4-based imaging. We determined that these CXCR4 antagonists were non-toxic and could be used to specifically label the CXCR4 receptor. Furthermore, IS4 and IS4-FAM inhibited CXCL12-stimulated cancer cell migration and Ca2+ release in both adherent and suspension cell lines with similar or improved potency as compared to two literature CXCR4 antagonists. Our results highlight the potential of IS4 and IS4-FAM as research tools and as potent CXCR4 antagonists for the prevention of metastasis.

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26 nM), AUC 7.30 ± 0.32 (IC50 50.43 nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15 nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48 h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.