Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

BACKGROUND: Stromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized. METHODS: The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed. RESULTS: Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts. CONCLUSION: These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis

Paternal Transmission of <i>X</i>-Linked Placental Dysplasia in Mouse Interspecific Hybrids

It has previously been shown that abnormal placental development, i.e., hyper- and hypoplasia, occurs in crosses and backcrosses between different mouse (Mus) species. These defects are caused mainly by abnormal growth of the spongiotrophoblast. The precise genetic basis for these placental malformations has not been determined. However, a locus that contributes to the abnormal development (Ihpd: interspecific hybrid placental dysplasia) has been mapped to the X chromosome. The X-chromosomal location of Ihpd and its site of action, that is the spongiotrophoblast, mean that normally only the maternally inherited Ihpd locus is active even in female fetuses. However, by making use of the X-chromosomal inversion In(X)1H, we have produced interspecific hybrid Xp0, in which the active X chromosome was inherited from Mus macedonicus males. In contrast to XX female and XY male conceptuses from this cross, which have hypoplastic placentas, the Xp0 female conceptuses have hyperplastic placentas. This finding supports the view that it is expression of the M. macedonicus Ihpd locus in the spongiotrophoblast that leads to hyperplasia due to an abnormal interaction with M. musculus autosomal loci.</jats:p

Abstract A217: Comparison of long-term pharmacodynamic actions of the synthetic small molecule HSP90 inhibitor AT13387 in multiple xenograft models

Abstract AT13387 is a novel small molecule inhibitor of HSP90. a member of a family of molecular chaperones. Previously we highlighted an association between the high affinity binding of AT13387 to the N-terminal ATPase domain of HSP90 and the duration of target inhibition in tumor cell lines in vitro. Further, AT13387 was shown to inhibit HSP90 and deplete client proteins in tumor xenografts longer than other, lower affinity inhibitors in the class. Here we have expanded the investigation to a wider number of tumor cell lines and to in vivo xenograft models and demonstrate that AT13387 has an extended pharmacodynamic action in tumors compared to other HSP90 inhibitors. We reason that the cumulative effects of these properties allow for less frequent dosing thus maximising efficacy whilst minimising systemic exposure and the potential for side effects. This study reports extended inhibition of HSP90 by AT13387 in a wider range of tumor cell lines in vitro. A 24hr exposure of A375 (melanoma) cells to AT13387 suppressed the expression of client proteins for 72 hrs or more. However in other cell lines such as NCI-H1975 (lung) and BT474 (breast), the suppression of client proteins by AT13387 was found to last in excess of 7 days. The pharmacodynamic action of AT13387 in vivo has been compared with that of 17-AAG and SNX-5422 in A375 and NCI-H1975 xenografts in nude mice. Following a single dose of each agent, we have investigated and compared the time course of the suppression of levels of several client proteins (e.g. AKT, CDK4) and the phosphorylation of key growth/survival signalling components (e.g. pERK, pS6, pAKT). These effects were rapidly induced in tumors following treatment with AT13387 and levels remained suppressed for up to 96 hrs. The durability of the AT13387 effects was significantly greater than for the other competitor compounds. Investigation of tumor growth in these models demonstrated that the longer pharmacodynamic action of AT13387 ensured that efficacy could be maintained on a once weekly schedule, whereas such a schedule for the other agents resulted in a significant loss of their anti-tumor effects. These data provide further support for the potential benefit of long acting HSP90 inhibitors as a way of maintaining anti-tumor effects whilst minimising potential for undesirable effects associated with systemic exposure. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A217.</jats:p

AT9283, a Potent Inhibitor of JAK2, Is Active in JAK2 V617F Myeloproliferative Disease Models.

Abstract AT9283 is a potent inhibitor of JAK2, JAK3, mutant Abl kinase (T315IAbl) and Aurora kinases A and B all of which have an IC50 &lt;5nM. The compound is currently in early phase clinical development for hematological malignancies. Multitargeted kinase inhibitors may be of particular value as anti-proliferative agents as a consequence of their ability to inhibit several signalling pathways simultaneously. Many of the kinases targeted by AT9283 lie in signalling pathways activated by oncogenes and may contribute in a positive way to the anti-tumour action of the compound. Here we describe the characterisation of the anti-tumour effects of AT9283 in models of JAK2-dependent disease The JAK2 V617F mutation has been identified in more than 95% of patients with Polycythemia vera and in 50 to 60% of patients with Essential Thrombocythemia and myelofibrosis. JAK2 is a key modulator of cytokine signalling, transducing signals from cell surface receptors via the JAK/STAT pathway. The point mutation renders the kinase constitutively active and induces cytokine-independent proliferation of cell lines harboring mutated JAK2. AT9283 was profiled against two cell lines harboring JAK2 V617F (HEL, SET-2) as well as two lines with a dependence on cytokine signalling through wild type endogenous JAK2 for survival (BA/F3 wt, TF-1, stimulated with IL3). These lines were compared with two additional leukaemia cell lines not dependent upon JAK signalling for survival, K562 (Ph+CML) and HL60 (N-Ras PML). In each case the dominant effect of the compound is to inhibit cell growth through JAK inhibition where the survival of the cell line depends on signalling through the JAK2 pathway. In contrast the cell lines less dependent on JAK2 for survival show a polyploid phenotype indicative of Aurora kinase inhibition. In HEL cells AT9283 inhibits phosphorylation of JAK2 pathway markers such as phospho-STAT5 (Tyr694) at 100nM, a dose consistent with concentrations required to inhibit the proliferation of these cells. Consequently, the cell cycle profiles generated when either K562 or HL60 cells are treated with AT9283 suggest that endoreduplication derived from Aurora inhibition is the dominant phenotype. In a JAK driven system the dominant phenotype observed is induction of apoptosis via inhibition of JAK. In an in vivo model, administration of a single dose of AT9283 to the JAK-dependent HEL xenograft, inhibited phosphorylation of STAT5 indicating JAK2 inhibition in the tumour tissue and an efficacy study demonstrated growth inhibitory effects of 9283 in this model. Finally the activity of AT9283 was tested in primary assays using cells taken either from healthy volunteers or patients diagnosed with MPD. These data together support further clinical investigation of the compound in patients with a myeloproliferative disorder.</jats:p

Fragment-based discovery of the pyrazol-4-yl urea (AT9283), a multitargeted kinase inhibitor with potent aurora kinase activity

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC50 ≈ 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials

AT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I tria