Divergent regulation of basement membrane trafficking by human macrophages and cancer cells.

Macrophages and cancer cells populations are posited to navigate basement membrane barriers by either mobilizing proteolytic enzymes or deploying mechanical forces. Nevertheless, the relative roles, or identity, of the proteinase -dependent or -independent mechanisms used by macrophages versus cancer cells to transmigrate basement membrane barriers harboring physiologically-relevant covalent crosslinks remains ill-defined. Herein, both macrophages and cancer cells are shown to mobilize membrane-anchored matrix metalloproteinases to proteolytically remodel native basement membranes isolated from murine tissues while infiltrating the underlying interstitial matrix ex vivo. In the absence of proteolytic activity, however, only macrophages deploy actomyosin-generated forces to transmigrate basement membrane pores, thereby providing the cells with proteinase-independent access to the interstitial matrix while simultaneously exerting global effects on the macrophage transcriptome. By contrast, cancer cell invasive activity is reliant on metalloproteinase activity and neither mechanical force nor changes in nuclear rigidity rescue basement membrane transmigration. These studies identify membrane-anchored matrix metalloproteinases as key proteolytic effectors of basement membrane remodeling by macrophages and cancer cells while also defining the divergent invasive strategies used by normal and neoplastic cells to traverse native tissue barriers.

Blocking downstream signaling pathways in the context of HDAC inhibition promotes apoptosis preferentially in cells harboring mutant Ras.

We previously demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in a series of romidepsin-selected T-cell lymphoma cell lines as a mechanism of resistance to the histone deacetylase inhibitor (HDI), romidepsin. As Ras mutation leads to activation of both the MAPK and the phosphoinositide 3-kinase (PI3K) pathway, we examined whether combining romidepsin with small molecule pathway inhibitors would lead to increased apoptosis in cancers harboring Ras mutations. We treated 18 Ras mutant or wild-type cell lines with romidepsin in the presence of a MEK inhibitor (PD-0325901) and/or an AKT inhibitor (MK-2206) and examined apoptosis by flow cytometry. A short-term treatment schedule of romidepsin (25 ng/ml for 6 h) was used to more closely model clinical administration. Romidepsin in combination with a MEK and an AKT inhibitor induced apoptosis preferentially in cells harboring mutant versus wild-type Ras (69.1% vs. 21.1%, p < 0.0001). Similar results were found in a subset of cell lines when belinostat was combined with the MEK and AKT inhibitors and when romidepsin was combined with the dual extracellular signaling-related kinase (ERK)/PI3K inhibitor, D-87503, which inhibited both the MAPK and PI3K pathways at 5-10 µM. The observed apoptosis was caspase-dependent and required Bak and Bax expression. Cells with wild-type or mutant Ras treated with romidepsin alone or in combination with the MEK inhibitor displayed increased expression of proapoptotic Bim. We thus conclude that cancers bearing Ras mutations, such as pancreatic cancer, can be targeted by the combination of an HDI and a dual inhibitor of the MAPK and PI3K pathways.

Loss of the proteins Bak and Bax prevents apoptosis mediated by histone deacetylase inhibitors.

Burkitt lymphoma is characterized by deregulation of c-myc, and therapies targeting c-myc are under investigation as treatments. Histone deacetylase inhibitors are known to abrogate c-myc expression, leading us to examine their effect in a series of Burkitt lymphoma cell lines. While treatment with romidepsin, panobinostat, vorinostat, or belinostat for 48 h resulted in complete cell death in the Ramos and ST486 lines, CA46 and DG75 cells were resistant. In parallel studies, CA46 and DG75 cells were also insensitive to 48 h treatment with the Aurora kinase inhibitors (AKIs) MLN8237 (alisertib), VX-680 (tozasertib), or ZM447439. Bax knockdown is known to lead to HDI resistance, and we found that loss of Bax or both Bak and Bax correlated with resistance to both AKIs and HDIs in the Burkitt cell lines. As proof-of-concept to evaluate the contribution of Bax and Bak to HDI-mediated apoptosis, we found that apoptosis was unaffected in HCT-116 colon carcinoma cells lacking Bak, blunted in cells lacking Bax, and nearly completely abrogated in cells lacking both Bak and Bax compared with wild-type cells. To explore potential clinical variations in Bak and Bax expression, a series of samples from 16 patients diagnosed with Burkitt lymphoma was examined. While the majority of samples were positive for both Bak and Bax, some (3/16) expressed low levels of both proteins. We thus conclude that HDI-mediated and AKI-mediated apoptosis requires mitochondrial engagement, and that baseline Bax and Bak expression may serve as biomarkers for patients with Burkitt lymphoma likely to respond to HDI treatment.

Becatecarin (rebeccamycin analog, NSC 655649) is a transport substrate and induces expression of the ATP-binding cassette transporter, ABCG2, in lung carcinoma cells.

PURPOSE: ABCG2 overexpression has been linked to resistance to topoisomerase inhibitors, leading us to examine the potential interaction between ABCG2 and becatecarin. METHODS: Interaction with ABCG2 was determined by ATPase assay, competition of [(125)I]iodoarylazidoprazosin (IAAP) photolabeling and flow cytometry. Cellular resistance was measured in 4-day cytotoxicity assays. ABCG2 expression was measured by fluorescent-substrate transport assays and immunoblot. RESULTS: Becatecarin competed [(125)I]-IAAP labeling of ABCG2, stimulated ATPase activity and, at concentrations greater than 10 microM, inhibited ABCG2-mediated transport. Becatecarin-selected A549 Bec150 lung carcinoma cells were 3.1-, 15-, 8-, and 6.8-fold resistant to becatecarin, mitoxantrone, SN-38 and topotecan, respectively. A549 Bec150 cells transported the ABCG2 substrates pheophorbide a, mitoxantrone and BODIPY-prazosin and displayed increased staining with the anti-ABCG2 antibody 5D3 compared to parental cells. Increased ABCG2 expression was confirmed by immunoblot. CONCLUSIONS: Our results suggest that becatecarin is transported by ABCG2 and can induce ABCG2 expression in cancer cells.