LSD1 activates a lethal prostate cancer gene network independently of its demethylase function.

Medical castration that interferes with androgen receptor (AR) function is the principal treatment for advanced prostate cancer. However, clinical progression is universal, and tumors with AR-independent resistance mechanisms appear to be increasing in frequency. Consequently, there is an urgent need to develop new treatments targeting molecular pathways enriched in lethal prostate cancer. Lysine-specific demethylase 1 (LSD1) is a histone demethylase and an important regulator of gene expression. Here, we show that LSD1 promotes the survival of prostate cancer cells, including those that are castration-resistant, independently of its demethylase function and of the AR. Importantly, this effect is explained in part by activation of a lethal prostate cancer gene network in collaboration with LSD1's binding protein, ZNF217. Finally, that a small-molecule LSD1 inhibitor-SP-2509-blocks important demethylase-independent functions and suppresses castration-resistant prostate cancer cell viability demonstrates the potential of LSD1 inhibition in this disease.

Comparative mechanisms of PAH toxicity by benzo[a]pyrene and dibenzo[def,p]chrysene in primary human bronchial epithelial cells cultured at air-liquid interface.

Current assumption for assessing carcinogenic risk of polycyclic aromatic hydrocarbons (PAHs) is that they function through a common mechanism of action; however, recent studies demonstrate that PAHs can act through unique mechanisms potentially contributing to cancer outcomes in a non-additive manner. Using a primary human 3D bronchial epithelial culture (HBEC) model, we assessed potential differences in mechanism of toxicity for two PAHs, benzo[a]pyrene (BAP) and dibenzo[def,p]chrysene (DBC), compared to a complex PAH mixture based on short-term biosignatures identified from transcriptional profiling. Differentiated bronchial epithelial cells were treated with BAP (100-500 µg/ml), DBC (10 µg/ml), and coal tar extract (CTE 500-1500 µg/ml, SRM1597a) for 48 h and gene expression was measured by RNA sequencing or quantitative PCR. Comparison of BAP and DBC gene signatures showed that the majority of genes (~60%) were uniquely regulated by treatment, including signaling pathways for inflammation and DNA damage by DBC and processes for cell cycle, hypoxia and oxidative stress by BAP. Specifically, BAP upregulated targets of AhR, NRF2, and KLF4, while DBC downregulated these same targets, suggesting a chemical-specific pattern in transcriptional regulation involved in antioxidant response, potentially contributing to differences in PAH potency. Other processes were regulated in common by all PAH treatments, BAP, DBC and CTE, including downregulation of genes involved in cell adhesion and reduced functional measurements of barrier integrity. This work supports prior in vivo studies and demonstrates the utility of profiling short-term biosignatures in an organotypic 3D model to identify mechanisms linked to carcinogenic risk of PAHs in humans.

Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470.

Angiogenesis is crucial for tumor growth. Angiogenesis inhibitors, such as O-(chloracetyl-carbamoyl) fumagillol (TNP-470), are thus emerging as a new class of anticancer drugs. In clinical trials, TNP-470 slowed tumor growth in patients with metastatic cancer. However, at higher doses necessary for tumor regression, many patients experienced neurotoxicity. We therefore synthesized and characterized a water-soluble conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, Gly-Phe-Leu-Gly linker and TNP-470. This conjugate accumulated selectively in tumor vessels because of the enhanced permeability and retention (EPR) effect. HPMA copolymer-TNP-470 substantially enhanced and prolonged the activity of TNP-470 in vivo in tumor and hepatectomy models. Polymer conjugation prevented TNP-470 from crossing the blood-brain barrier (BBB) and decreased its accumulation in normal organs, thereby avoiding drug-related toxicities. Treatment with TNP-470 caused weight loss and neurotoxic effects in mice, whereas treatment with the conjugate did not. This new approach for targeting angiogenesis inhibitors specifically to the tumor vasculature may provide a new strategy for the rational design of cancer therapies.

Alternative splicing of LSD1+8a in neuroendocrine prostate cancer is mediated by SRRM4.

Neuroendocrine prostate cancer (NEPC) is the most virulent form of prostate cancer. Importantly, our recent work examining metastatic biopsy samples demonstrates NEPC is increasing in frequency. In contrast to prostate adenocarcinomas that express a luminal gene expression program, NEPC tumors express a neuronal gene expression program. Despite this distinction, the diagnosis of NEPC is often challenging, demonstrating an urgent need to identify new biomarkers and therapeutic targets. Our prior work demonstrated that the histone demethylase LSD1 (KDM1A) is important for survival of prostate adenocarcinomas, but little was known about LSD1's role in NEPC. Recently, a neural-specific transcript variant of LSD1-LSD1+8a-was discovered and demonstrated to activate neuronal gene expression in neural cells. The splicing factor SRRM4 was previously shown to promote LSD1+8a splicing in neuronal cells, and SRRM4 promotes NEPC differentiation and cell survival. Therefore, we sought to determine if LSD1+8a might play a role in NEPC and whether LSD1+8a splicing was linked to SRRM4. To investigate a potential role for LSD1+8a in NEPC, we examined a panel of prostate adenocarcinoma and NEPC patient-derived xenografts and metastatic biopsies. LSD1+8a was expressed exclusively in NEPC samples and correlated significantly with elevated expression of SRRM4. Using SRRM4-overexpressing cell lines, we determined that SRRM4 mediates alternative splicing of LSD1+8a. Finally, using gain of function studies, we confirmed that LSD1+8a and SRRM4 co-regulate target genes distinct from canonical LSD1. Our findings suggest further study of the interplay between SRRM4 and LSD1+8a and mechanisms by which LSD1+8a regulates gene expression in NEPC is warranted.

Nonsteroidal antiinflammatory drugs differentially suppress endometriosis in a murine model.

OBJECTIVE: To determine whether nonsteroidal antiinflammatory drugs (NSAIDs) affect the establishment and progression of endometriotic lesions in a murine model. DESIGN: Pharmacologic intervention in a surgically induced murine model of abdominal/peritoneal endometriosis. SETTING: Animal research facility. PATIENT(S): Eight-week-old, female C57BL/6 mice. INTERVENTION(S): After implantation of autologous endometrium, mice were randomized into groups and treated with one of several NSAIDs or the vehicle-matched control for 4 weeks. MAIN OUTCOME MEASURE(S): Establishment, growth, and total burden of endometriotic lesions. RESULT(S): The NSAIDs differentially inhibited lesion establishment and growth, resulting in significantly reduced disease burden. Compared with controls (5.7 +/- 2.3 mm(2)), lesion burden was reduced by celecoxib (1.3 +/- 1.2 mm(2)), indomethacin (1.4 +/- 1.4 mm(2)), naproxen (2.7 +/- 1.2 mm(2)), sulindac (3.1 +/- 1.5 mm(2)), rofecoxib (3.4 +/- 3.0 mm(2)), and ibuprofen (4.1 +/- 1.4 mm(2)). In contrast, aspirin (5.9 +/- 1.2 mm(2)) had no statistically significant effect. Uninterrupted estrus cycling was confirmed by vaginal exams and smears in celecoxib-treated mice. CONCLUSION(S): Chronic administration of certain NSAIDs limits the progression of endometriosis in this murine model. The data suggest that NSAID selection in the treatment of endometriosis should be extended beyond pain management to maximize the inhibitory effect on disease burden.

Short synthetic endostatin peptides inhibit endothelial migration in vitro and endometriosis in a mouse model.

OBJECTIVE: To determine the active peptide regions inside the angiogenesis inhibitor endostatin that can inhibit endothelial migration in vitro and also inhibit endometriosis in a mouse model. DESIGN: Pharmacologic intervention in a surgically induced mouse model of endometriosis and endothelial migration assay. SETTING: Animal research and laboratory facility. SUBJECT(S): Eight-week-old, female C57BL/6 mice and human microvascular endothelial cells. INTERVENTION(S): Eight overlapping synthetic peptides were tested for inhibitory potential on endothelial migration in vitro. The peptides with significant activity then were given for 4 weeks to mice after implantation of autologous endometrium. MAIN OUTCOME MEASURE(S): Inhibition of vascular endothelial growth factor-induced endothelial migration for in vitro studies. In vivo studies examined the growth rate of endometriotic lesions after 4 weeks of treatment, as well as the effect on estrous cycling and ovulation as assessed by corpus luteum formation. RESULT(S): The N-terminal mP-1 peptide and the internal mP-6 peptide inhibited endothelial migration in a dose-dependent manner. Additionally, both synthetic peptides suppressed growth of endometriotic lesions significantly in vivo. However, estrous cycling and corpus luteum formation were normal in both groups. CONCLUSION(S): Short endostatin fragments may be promising as a new, nontoxic therapeutic strategy for the treatment of endometriosis without inhibition of normal estrous cycles.

Endostatin inhibits the growth of endometriotic lesions but does not affect fertility.

OBJECTIVE: To determine whether endometriosis can be treated with the angiogenesis inhibitor endostatin and the effect of this treatment on fertility and reproduction. DESIGN: Pharmacologic intervention in a surgically induced model of endometriosis and in female mice undergoing mating. SETTING: Animal research facility. ANIMAL(S): Eight-week-old, female C57BL/6 and SCID mice. INTERVENTION(S): After implantation of autologous endometrium, mice received endostatin or the vehicle-matched control for 4 weeks. For the reproductive function study, mice receiving endostatin or vehicle were mated and reproductive functions were observed. MAIN OUTCOME MEASURE(S): Growth of endometriotic lesions after 4 weeks of treatment; estrous cycling, corpus luteum formation, serum hormone levels, and mating time as fertility measures; and pregnancy rates, length of pregnancy, fetal vitality, number, and outcome of litter as reproductive measures. RESULT(S): Endostatin suppressed the growth of endometriotic lesions by 47% compared with controls. Estrous cycling and corpus luteum formation were normal in both groups. Female mice receiving endostatin were as fertile as mice receiving vehicle, had normal pregnancies, and delivered the same number of pups. The offspring were healthy without teratogenic stigmata and reproduced normally themselves. CONCLUSION(S): Antiangiogenic therapy with endostatin may present a promising novel, nontoxic therapeutic option for patients with endometriosis.