Discovery of the Selective and Orally Available Galectin-1 Inhibitor GB1908 as a Potential Treatment for Lung Cancer.

We have previously described a new series of selective and orally available galectin-1 inhibitors resulting in the thiazole-containing glycomimetic GB1490. Here, we show that the introduction of polar substituents to the thiazole ring results in galectin-1-specific compounds with low nM affinities. X-ray structural analysis of a new ligand-galectin-1 complex shows changes in the binding mode and ligand-protein hydrogen bond interactions compared to the GB1490-galectin-1 complex. These new high affinity ligands were further optimized with respect to affinity and ADME properties resulting in the galectin-1-selective GB1908 (Kd galectin-1/3 0.057/6.0 µM). In vitro GB1908 inhibited galectin-1-induced apoptosis in Jurkat cells (IC50 = 850 nM). Pharmacokinetic experiments in mice revealed that a dose of 30 mg/kg b.i.d. results in free levels of GB1908 in plasma over galectin-1 Kd for 24 h. GB1908 dosed with this regimen reduced the growth of primary lung tumor LL/2 in a syngeneic mouse model.

Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression.

We show that the centrosome- and microtubule-regulating protein γ-tubulin interacts with E2 promoter binding factors (E2Fs) to modulate E2F transcriptional activity and thereby control cell cycle progression. γ-Tubulin contains a C-terminal signal that results in its translocation to the nucleus during late G(1) to early S phase. γ-Tubulin mutants showed that the C terminus interacts with the transcription factor E2F1 and that the E2F1-γ-tubulin complex is formed during the G(1)/S transition, when E2F1 is transcriptionally active. Furthermore, E2F transcriptional activity is altered by reduced expression of γ-tubulin or by complex formation between γ-tubulin and E2F1, E2F2, or E2F3, but not E2F6. In addition, the γ-tubulin C terminus encodes a DNA-binding domain that interacts with E2F-regulated promoters, resulting in γ-tubulin-mediated transient activation of E2Fs. Thus, we report a novel mechanism regulating the activity of E2Fs, which can help explain how these proteins affect cell cycle progression in mammalian cells.

Germline RBBP8 variants associated with early-onset breast cancer compromise replication fork stability.

Haploinsufficiency of factors governing genome stability underlies hereditary breast and ovarian cancer. One significant pathway that is disabled as a result is homologous recombination repair (HRR). With the aim of identifying new candidate genes, we examined early-onset breast cancer patients negative for BRCA1 and BRCA2 pathogenic variants. Here, we focused on CtIP (RBBP8 gene), which mediates HRR through the end resection of DNA double-strand breaks (DSBs). Notably, these patients exhibited a number of rare germline RBBP8 variants. Functional analysis revealed that these variants did not affect DNA DSB end resection efficiency. However, expression of a subset of variants led to deleterious nucleolytic degradation of stalled DNA replication forks in a manner similar to that of cells lacking BRCA1 or BRCA2. In contrast to BRCA1 and BRCA2, CtIP deficiency promoted the helicase-driven destabilization of RAD51 nucleofilaments at damaged DNA replication forks. Taken together, our work identifies CtIP as a critical regulator of DNA replication fork integrity, which, when compromised, may predispose to the development of early-onset breast cancer.

BAP1 induces cell death via interaction with 14-3-3 in neuroblastoma.

BRCA1-associated protein 1 (BAP1) is a nuclear deubiquitinating enzyme that is associated with multiprotein complexes that regulate key cellular pathways, including cell cycle, cellular differentiation, cell death, and the DNA damage response. In this study, we found that the reduced expression of BAP1 pro6motes the survival of neuroblastoma cells, and restoring the levels of BAP1 in these cells facilitated a delay in S and G2/M phase of the cell cycle, as well as cell apoptosis. The mechanism that BAP1 induces cell death is mediated via an interaction with 14-3-3 protein. The association between BAP1 and 14-3-3 protein releases the apoptotic inducer protein Bax from 14-3-3 and promotes cell death through the intrinsic apoptosis pathway. Xenograft studies confirmed that the expression of BAP1 reduces tumor growth and progression in vivo by lowering the levels of pro-survival factors such as Bcl-2, which in turn diminish the survival potential of the tumor cells. Patient data analyses confirmed the finding that the high-BAP1 mRNA expression correlates with a better clinical outcome. In summary, our study uncovers a new mechanism for BAP1 in the regulation of cell apoptosis in neuroblastoma cells.

Deubiquitination of γ-tubulin by BAP1 prevents chromosome instability in breast cancer cells.

Microtubule nucleation requires the γ-tubulin ring complex, and during the M-phase (mitosis) this complex accumulates at the centrosome to support mitotic spindle formation. The posttranslational modification of γ-tubulin through ubiquitination is vital for regulating microtubule nucleation and centrosome duplication. Blocking the BRCA1/BARD1-dependent ubiquitination of γ-tubulin causes centrosome amplification. In the current study, we identified BRCA1-associated protein-1 (BAP1) as a deubiquitination enzyme for γ-tubulin. BAP1 was downregulated in metastatic adenocarcinoma breast cell lines compared with noncancerous human breast epithelial cells. Furthermore, low expression of BAP1 was associated with reduced overall survival of patients with breast cancer. Reduced expression of BAP1 in breast cancer cell lines was associated with mitotic abnormalities. Importantly, rescue experiments including expression of full length but not the catalytic mutant of BAP1 reduced ubiquitination of γ-tubulin and prevented mitotic defects. Our study uncovers a new mechanism for BAP1 involved in deubiquitination of γ-tubulin, which is required to prevent abnormal mitotic spindle formation and genome instability.

Association of nuclear-localized Nemo-like kinase with heat-shock protein 27 inhibits apoptosis in human breast cancer cells.

Nemo-like kinase (NLK), a proline-directed serine/threonine kinase regulated by phosphorylation, can be localized in the cytosol or in the nucleus. Whether the localization of NLK can affect cell survival or cell apoptosis is yet to be disclosed. In the present study we found that NLK was mainly localized in the nuclei of breast cancer cells, in contrast to a cytosolic localization in non-cancerous breast epithelial cells. The nuclear localization of NLK was mediated through direct interaction with Heat shock protein 27 (HSP27) which further protected cancer cells from apoptosis. The present study provides evidence of a novel mechanism by which HSP27 recognizes NLK in the breast cancer cells and prevents NLK-mediated cell apoptosis.