Prostate cancer affects 1 in 6 men and is the second most common cause of cancer-related death. Since the majority of prostate cancers critically depend on androgens for growth, androgen deprivation therapy is a frontline treatment for advanced prostate cancer. The majority of prostate tumors initially respond to androgen deprivation therapy, but eventually the disease often progresses to castration-resistant prostate cancer. However, the current therapies that only reduce androgen produced in the testis do not affect androgen produced by adrenal glands can also produce androgens. This extra-testicular androgen production is thought to be a key factor in the development of castration resistant prostate cancer. CYP17 is a P450 enzyme that catalyzes the last step of androgen biosynthesis in both the testis and the adrenals. Inhibition of CYP17 therefore completely blocks androgen production and it is thus thought to be more effective to prevent prostate cancer progression than current therapies. Angion Biomedica has identified a promising series of proprietary non-steroidal CYP17 inhibitors. Our lead compound is potent, orally bioavailable, and significantly reduces serum testosterone levels in mice. Treatment of rats and mice with our lead CYP17 inhibitor significantly reduces the weights of androgen dependent organs. In two mouse xenograft models of androgen-dependent tumor growth our lead CYP17 inhibitor markedly reduces primary tumor growth, serum testosterone and PSA levels. Angion is currently further evaluating the therapeutic potential of this series of compounds as a possible therapy for castration resistant prostate cancer. Citation Format: Bert Oehlen, Siobhan McCormack, Gaifeng Ma, Dong Sung Lim, Bijoy Panicker, Itzhak D. Goldberg. Characterization of a new non-steroidal small molecule inhibitor of CYP17 for castration-resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2013-5560

Abstract Retinoids, such as the prototypical retinoid all trans retinoic acid (ATRA), have been used successfully in the treatment of acute promyelocytic leukemia (APL) patients. They have also been tested for efficacy in solid tumors, including breast cancer. Despite promising preclinical data, the clinical success of using ATRA for breast cancer patients has been limited so far. We hypothesize that this is largely due to three factors: (1) a lack of understanding of the patient population that is likely to respond to retinoid therapy, (2) a significant induction of retinoic acid metabolism with retinoid therapy, resulting in sub-optimal retinoid exposure in tumors and (3) the narrow therapeutic index of ATRA. Several recent studies indicate that breast cancer cells that express the Retinoic Acid Receptor Alpha (RARA) are more likely to respond to retinoic acid than cells without RARA expression. This provides a rationale for selection of patients that are likely to benefit from retinoid therapy in future clinical trials. The cytochrome P450 enzyme CYP26 is the key enzyme involved in ATRA metabolism. CYP26A1 expression is elevated in breast cancer tissue compared to normal tissue and CYP26A1 expression is negatively correlated with patient survival. Angion has identified orally bioavailable small molecule inhibitors of CYP26. Angion's lead inhibitor blocks retinoid metabolism in tumor cells and enhances serum and tumor retinoic acid levels. The compound shows robust single agent efficacy in several murine breast cancer xenograft models. By elevating endogenous serum and tumor retinoic acid levels in a physiological range, CYP26 inhibition therapy is likely to result in a better therapeutic index than systemic retinoid therapy. The identification of orally bioavailable, potent, and selective CYP26 inhibitors eliminates two major drawbacks of systemic retinoid therapy, and– in combination with new insights in selecting patients that are more likely to respond to retinoid therapy– may provide a new impetus for evaluation of retinoic acid signaling modulators as a treatment for breast cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B257. Citation Format: Bert Oehlen, Gaifeng Ma, Siobhan McCormack, Dong Sung Lim, Jim Tarrant, Xiaokang Zhu, Bijoy Panicker, Itzhak D. Goldberg. Identification of new CYP26 inhibitors with efficacy in breast cancer xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B257.

Exisulind (sulindac sulfone) and three highly potent derivatives, OSI-461 (CP461), OSIP486823 (CP248), and OSIP487703, inhibit growth and induce apoptosis in SW480 human colon cancer cells, with IC(50)s of 200, 2, 0.1, and 0.003 micromol/L, respectively. The latter three compounds, but not exisulind, induce marked M-phase cell cycle arrest in these cells. This effect seems to be independent of the known ability of these compounds to cause activation of protein kinase G. When tested at twice their IC(50) concentration for growth inhibition, OSI-461, OSIP486823, and OSIP487703 cause depolymerization of microtubules in interphase cells, inhibit spindle formation in mitotic cells, and induce multinucleated cells. In vitro tubulin polymerization assays indicate that all three compounds interact with tubulin directly to cause microtubule depolymerization and/or inhibit de novo tubulin polymerization. These results suggest that the dual effects of OSI-461, OSIP486823, and OSIP487703 on impairment of microtubule functions and protein kinase G activation may explain the potent antiproliferative and apoptotic effects of these compounds in cancer cells.

Proc Amer Assoc Cancer Res, Volume 46, 2005 2330 There is increasing evidence that activation of the cGMP-dependent enzyme protein kinase G (PKG) can play an important role in inhibiting cell proliferation and inducing apoptosis. The intracellular level of cGMP is regulated through a dynamic balance between its rate of synthesis by guanylyl cyclases (GCs) and its degradation by specific cGMP-phosphodiesterases (PDEs), especially PDEs 2 and 5. In previous studies we found that cGMP-PDE inhibitors, a GC activator, and constitutively activated mutants of PKG Iβ induced apoptosis in SW480 human colon cancer cells (Deguchi et al., Mol. Cancer Ther. 1, 803-809, 2002, and Cancer Res. 64, 3966-3973, 2004). However, the actual profile of components of the cGMP/PKG signaling pathway in human cancers has not been previously studied in detail. In this study, we examined by western blot analysis cellular levels of PDE5, PKG Iβ and phospho-VASP (vasodilator-stimulated phosphoprotein), a marker of PKG activation, in 19 pairs of primary human colon carcinomas and paired adjacent normal colonic mucosa. PKG Iβ was decreased in 54%, PDE5 was increased in 58%, and phospho-VASP was decreased in 89% of the colon tumors. We also did similar assays in several cell lines. When compared to the normal human fetal colon cell line CCD841CoN cells, PDE5 was increased and phospho-VASP was decreased in the colon cancer cell lines SW480, SW620, and DLD1. When compared to MCF-10F normal human mammary epithelial cells, MCF7 human breast cancer cells also displayed an increase in expression of PDE5 and decreased levels of PKG Iβ and phospho-VASP. Furthermore, ras- and raf-transformed Rat6 fibroblasts displayed decreased levels of PKG Iβ and increased levels of PDE5 when compared with the parental Rat6 cells. Taken together, these findings suggest that down-regulation of cGMP/PKG-mediated signaling pathways often occurs during tumorigenesis and cell transformation. These results are consistent with previous evidence indicating that agents that increase the activation of PKG can inhibit growth and induce apoptosis in cancer cells, and provide a rationale for the further development of such agents as a novel strategy for cancer therapy.

Proc Amer Assoc Cancer Res, Volume 46, 20054499 Lysophosphatidic acid (LPA) is a well characterized growth and survival factor for cancer cells in culture. This study details the identification of two G-protein coupled receptors, OSGPR78 and OSGPR114 as novel lysophosphatidic acid (LPA) receptors. OSGPR78 and OSGPR114, when expressed in a yeast-based reporter assay, are activated by multiple analogs of LPA, including myristoyl, palmitoyl, oleoyl, stearoyl and polyunsaturated LPA analogs. Similarly, when expressed in mammalian cells, the receptors also respond to the same LPA analogs. Pertussis toxin sensitivity of the effects of LPA activation of OSGPR114 expressed in CHO cells suggests a coupling to Gi/o proteins. Amino acid homologies of OSGPR114 and OSGPR78 demonstrate no obvious similarity with the known LPA G-protein coupled receptors LPA1-3. The expression of these novel LPA receptors in normal human tissues, cancer cell lines and in tumor samples has been characterized using fluorogenic real-time PCR. In normal tissues, OSGPR114 was expressed at the highest levels in areas such as the lung, kidney, leukocytes and ovary, whereas OSGPR78 was found to be expressed at high levels in almost every tissue studied. Several cancer cell lines have been identified which express OSGPR78 or OSGPR114 as the predominant LPA receptor and both receptors are widely expressed in human tumor samples. Furthermore, in cell lines shown to express OSGPR114 or OSGPR78, small interfering RNA oligonucleotides (siRNA) designed to specifically knockdown OSGPR114 and OSGPR78 expression induced a growth-inhibitory and pro-apoptotic phenotype in cells maintained in full serum. In summary, we demonstrate that OSGPR78 and OSGPR114 are novel LPA activated G-protein coupled receptors. Initial evidence suggests that OSGPR114 and OSGPR78, like the known LPA receptors (LPA1-3 and gpr23), are involved in mediating the proliferation and survival effects of LPA on cancer cells in culture.

Recent studies indicate that the induction of apoptosis in human colon cancer cells by certain nonsteroidal antiinflammatory drugs involves increased expression of 15-LOX-1 and synthesis of its major product 13-S-hydroxyoctadecadienoic acid (13-S-HODE). Evidence was obtained that this occurs via a cyclooxygenase-2 (COX-2)-independent mechanism, but the actual mechanism of induction of 15-LOX-1 by these compounds is not known. There is extensive evidence that treatment of SW480 human colon cancer cells with sulindac sulfone (Exisulind, Aptosyn) or the related derivative OSI-461, both of which inhibit cyclic GMP (cGMP)-phosphodiesterases but lack COX-2 inhibitory activity, causes an increase in intracellular levels of cGMP, thus activating protein kinase G (PKG), which then activates pathways that lead to apoptosis. Therefore, in the present study, we examined the effects of various agents that cause increased cellular levels of cGMP on the expression of 15-LOX-1 in SW480 human colon cancer cells. Treatment of the cells with Exisulind, sulindac sulfide, OSI-461, the guanylyl cyclase activator YC-1, or the cell-permeable cGMP compound 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) caused an increase in cellular levels of 15-LOX-1. Exisulind, OSI-461, and 8-pCPT-cGMP also increased mRNA levels of 15-LOX-1, suggesting that the effects were at the level of transcription. The cGMP-phosphodiesterase inhibitors and YC-1 increased the production of 13-S-HODE, which is the linoleic acid metabolite of 15-LOX-1. Treatment of SW480 cells with the PKG inhibitor Rp-8-pCPT-cGMP blocked Exisulind-induced 15-LOX-1 expression. Furthermore, derivatives of SW480 cells that were engineered to stably overexpress wild-type PKG Ibeta displayed increased cellular levels of 15-LOX-1 when compared with vector control cells. Taken together, these results provide evidence that the cGMP/PKG pathway can play an important role in the induction of 15-LOX-1 expression by nonsteroidal antiinflammatory drugs and related agents.

As a key part of the mechanism which controls growth and division, cells are able to respond to a variety of intracellular and extracellular stimuli. Significant progress has been made in the understanding of the biochemical mechanisms underlying mating-factor signal transduction in Saccharomyces cerevisiae. Some of these mechanisms may be relevant to the regulation of other signal transduction pathways.