Ling Jong

Regulation of RARβ expression by RAR‐ and RXR‐selective retinoids in human lung cancer cell lines: Effect on growth inhibition and apoptosis induction

Retinoids regulate the growth and differentiation of human tracheobronchial epithelial cells. In this study, we investigated the effects of all‐trans‐retinoic acid (trans‐RA) and receptor class‐selective retinoids on the growth and apoptosis of human lung cancer cell lines. Trans‐RA significantly inhibited the growth of Calu‐6 and H460 cells, accompanied by induction of RA receptor (RAR)β expression. In contrast, it had little effect on the growth of H292, SK‐MES‐1 and H661 lung cancer cell lines, in which RARβ expression was not induced. Stable expression of RARβ in RARβ‐negative, trans‐RA‐resistant SK‐MES‐1 and H661 lung cancer cells led to recovery of trans‐RA‐induced growth inhibition, which occurred, however, only at low serum concentration. Using fluorescent microscopy and the terminal deoxyribonucleotidyl transferase (TdT) assay, we demonstrated that induction of apoptosis by trans‐RA contributed to its growth‐inhibitory effect in trans‐RA‐sensitive lung cancer cell lines. Analysis of RAR‐selective and retinoid X receptor (RXR)‐selective retinoids showed that activation of both RARs and RXRs could induce growth inhibition in trans‐RA‐sensitive lung cancer cells. Also, an additive synergistic effect on growth inhibition and RARβ induction was observed when cells were treated with combinations of RAR‐selective and RXR‐selective retinoids. Together, our results show that expression of RARβ plays a role in mediating retinoid response in lung cancer cells and that activation of RARs or RXRs contributes to induction of RARβ, growth inhibition and apoptosis by retinoids. Int. J. Cancer 75:88–95, 1998.© 1998 Wiley‐Liss, Inc.

Effects of receptor class- and subtype-selective retinoids and an apoptosis-inducing retinoid on the adherent growth of the NIH:OVCAR-3 ovarian cancer cell line in culture.

Comparison of the adherent growth inhibition of NIH:OVCAR-3 ovarian cancer cells by retinoid receptor class-selective and subtype-selective compounds with their receptor binding affinities and transcriptional activation activities indicated no correlation for RAR alpha and RAR gamma although both receptors are present. Retinoids that activated RXR alpha inhibited cell growth in the range as all-trans-retinoic acid and 9-cis-retinoic acid. The most potent inhibitor was 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), which has been found to inhibit breast and lung cancer and leukemia cell growth and induce cancer cell apoptosis through a pathway independent of the retinoid receptors.

Retinoic acid (RA) receptor transcriptional activation correlates with inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase (ODC) activity by retinoids: a potential role for trans-RA-induced ZBP-89 in ODC inhibition.

Evaluation of retinoic acid receptor (RAR) subtype–selective α and γ agonists and antagonists and a retinoid X receptor (RXR) class–selective agonist for efficacy at inhibiting both induction of ornithine decarboxylase (ODC) by the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in mouse epidermis and rat tracheal epithelial cells and the appearance of papillomas in mouse epidermis treated in the 2‐stage tumor initiation–promotion model indicated that (i) RXR class–selective transcriptional agonists, such as MM11246, were not involved in ODC inhibition; (ii) RAR‐selective agonists that induce gene transcription from RA‐responsive elements (RAREs) were active at low concentrations; (iii) RAR‐selective antagonists that bind RARs and inhibit AP‐1 activation on the collagenase promoter but do not activate RAREs to induce gene transcription were less effective inhibitors; and (iv) RARγ‐selective retinoid agonists were more effective inhibitors of TPA‐induced ODC activity than RARα‐selective agonists. These results suggest that RARE activation has a more important role in inhibition of ODC activity than RXR activation or AP‐1 inhibition and that RARγ‐selective agonists would be the most useful inhibitors of epithelial cell proliferation induced by tumor promoters. The natural retinoid all‐trans‐RA induced expression of transcription factor ZBP‐89, which represses activation of the GC box in the ODC promoter by the transcription factor Sp1. Int. J. Cancer 91:8–21, 2001.

Identification of a unique binding protein specific for a novel retinoid inducing cellular apoptosis.

The retinoid 6‐[3‐(1‐adamantyl)‐4‐hydroxyphenyl]‐2‐naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL‐60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9‐cis‐retinoic acid (9‐cis‐RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9‐cis‐RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA‐MB‐468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR‐signaling pathways. Int. J. Cancer 86:474–479, 2000.

4-[3-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)phenyl]benzoic acid and heterocyclic-bridged analogues are novel retinoic acid receptor subtype and retinoid X receptor α agonists

Aromatic retinoids having a meta-substituted aromatic ring bridge, such as 4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)phenyl]benzo ic acid and its 3,5-diaryl-substituted 4,5-dihydroisoxazole analogue, function as retinoid receptor panagonists by activating both retinoic acid and retinoid X receptors to induce gene transcription, and thereby provide novel scaffolds for retinoid drug development. Both classes of these ligand-inducible transcription factors are involved in mediating the inhibitory effects of retinoids on cancer cell growth.

sp2-bridged diaryl retinoids: effects of bridge-region substitution on retinoid X receptor (RXR) selectivity.

RXR class selectivity and RXR transcriptional activation activity compared to those for the retinoic acid receptor subtypes were enhanced on the 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylethenyl)be nzoic acid scaffold and its 3-methyl analogue by replacing their 1,1-ethenyl bridge by a 1,1-(2-methylpropenyl) or cyclopropylidenylmethylene group.

Synthetic retinoids and their usefulness in biology and medicine

The natural retinoids—all-trans-retinoic acid (trans-RA) and its 9-cis isomer (9-cis-RA)—and their synthetic analogs have important functions in regulating cell proliferation, differentiation, and morphogenesis. For these reasons, retinoids are successfully used as therapeutic agents to treat several proliferative diseases, such as acne, psoriasis, skin cancer, and acute promyleoblastic leukemia, and to prevent the recurrence of head-and-neck and certain skin cancers [1-3]. Because of their ability to modulate cell differentiation and proliferation, retinoids at therapeutically effective doses may have such toxic side effects on normal cells that their use is limited [4]. A continuing goal of numerous drug development programs has been to identify therapeutically effective retinoids that have reduced systemic side effects [5] Such an approach is possible because retinoid activities are regulated through their receptor proteins.

Abstract LB-344: MAP4K4 as a therapeutic target and novel biomarker for NSCLC

Introduction: Biomarker-guided personalized targeted therapies have improved lung cancer treatment; however, non-small cell lung cancer (NSCLC) targets such as KRAS — where activated mutations occur in ∼30% of cases — have so far eluded attempts at therapeutic targeting. There continues to be an urgent need to identify molecular mechanisms driving the disease and new targeted therapies. Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) overexpression promotes migration and invasion in several cancer types and correlates with poor prognosis in lung cancer. We investigated the role of MAP4K4 in NSCLC and tested whether MAP4K4 could serve as a biomarker on circulating tumor cells (CTCs) to monitor pre-metastatic and late stage disease. Methods: We used a novel class of highly selective small molecule inhibitor of MAP4K4, represented by SRI-28731, as a tool to further elucidate the role of MAP4K4 in NSCLC. We measured the expression of MAP4K4 and signaling changes following compound inhibition in lung cancer cell lines by immunoblotting and immunofluorescence. Clonogenic and wound healing assays were performed using our selective compound to assess proliferation and migration. To determine if MAP4K4 could be used as a biomarker in liquid biopsies we used FASTcell™, a non-enrichment optical scanning technology that enables identification and characterization of 1 tumor cell in 25 million blood cells. Results: We confirmed high expression of MAP4K4 in 4 out of 7 lung cancer cell lines, including A549 cells, and 3 of these harbored KRAS mutations. By clonogenic assay, A549 cell proliferation was inhibited by 51% and 99% with 2.5 nM and 5 nM of SRI-28731, respectively. In addition, lung cancer cell migration was impaired in the presence of 10 or 20 nM SRI-28731. Smad 1/5/8 phosphorylation was decreased in cells treated with the MAP4K4 inhibitor. Compound treated cells displayed a change in their actin cytoskeleton compared to DMSO treated cells. To explore whether MAP4K4 could be used as a biomarker on CTCs of metastatic NSCLC patients, we utilized the FASTcell™ platform and detected MAP4K4 expression on A549 cells mixed into normal blood. We are in the process of testing the assay with additional lung cancer cell lines and also testing MAP4K4 expression on CTCs from lung cancer patients. Conclusions: Our preliminary data suggest a role for MAP4K4 in proliferation, migration and signaling in lung cancer. Inhibition of MAP4K4 may be a valid therapeutic approach and the expression of MAP4K4 may serve as a biomarker in liquid biopsies. We showed that high levels of MAP4K4 in human lung cancer cell lines was concomitant to KRAS mutations, suggesting a possible correlation between the two markers. This novel biomarker could provide an opportunity to detect KRAS-positive cancers earlier and our new selective inhibitor could lead to life-saving, personalized treatment options for NSCLC cancer patients. Citation Format: Claire E. Repellin, Xiaohe Liu, Mary R. Stofega, Janey C.L. Snider, Zheng Ao, Ling Jong, Lidia C. Sambucetti. MAP4K4 as a therapeutic target and novel biomarker for NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-344.

Abstract 731: SRI-28731, a highly potent and selective MAP4K4 (HGK) inhibitor for cancer therapy

MAP4K4, a Ser/Thr kinase, was identified as an important pro-migratory kinase in an siRNA screen, targeting 5,234 human genes for modulators of tumor cell motility. MAP4K4 siRNA potently suppressed cell invasion and migration of multiple cancer cell lines, indicating a broad role in cell motility. There are no drugs in the clinic that are known to specifically target MAP4K4 for cancer therapy. We have successfully developed an orally active, highly effective and selective MAP4K4 inhibitor (SRI-28731) with potent in vitro and in vivo anticancer activity. SRI-28731 is more potent than Paclitaxel (Taxol) against most of the breast cancer cell lines tested. SRI-28731 exhibits more potent activities against triple negative (MDA-MB-231, BT549 and Hs578T) than estrogen-dependent (T47D and MCF-7) breast cancer cell lines, and its potency is positively correlated with MAP4K4 expression in cancer cell lines. SRI-28731 is also more potent than Docetaxel against both androgen-dependent (LNCaP) and -independent (PC-3 and DU-145) prostate cancer cell lines. In vitro mechanistic studies showed that SRI-28731 induced apoptosis and a time-dependent M phase arrest. Treatment with SRI-28731 (12.5, 25 and 50 mg/kg/day) caused a significant dose-dependent growth reduction of PC-3 tumors (30%, 61% and 88% growth inhibition, respectively), while Docetaxel at its MTD (7.5 mg/kg; Q3Dx2) produced only 10-15% growth inhibition. At the end of PC-3 tumor xenograft studies, we conducted an ex vivo invasion assay using PC-3 tumor cells isolated from tumor-bearing mice. SRI-28731 significantly reduced ex vivo tumor cell invasion by ∼80%. Pharmacokinetic studies showed that SRI-28731 could be detected in plasma up to 8 hours after oral dosing, and drug plasma concentrations remained above the IC50 values needed to inhibit prostate or breast cancer proliferation. To quantitatively define the kinase selectivity of SRI-28731, we tested the interaction of SRI-28731 with 456 kinase protein kinases (KINOMEscan), followed by in vitro pharmacology studies. Our data indicated that SRI-28731 is a highly selective Type-II MAP4K4 inhibitor. Type II kinase inhibitors bind to both the ATP site and an adjacent hydrophobic site exposed in the non-activated kinase state. Generally, type II inhibitors show higher selectivity for targets, and act primarily by locking the equilibrium switch between conformational states in a way that prevents kinase activation, rather than directly inhibiting it. Elevated MAP4K4 expression is strongly associated with higher rate of metastasis, and is regarded as an independent predictor of overall survival in cancer patients. Since MAP4K4 is overexpressed in many human cancer cell lines but is undetectable in non-transformed epithelial cells, targeting MAP4K4 may provide effective anti-metastatic therapy with limited side effects on normal tissues. Citation Format: Chih-Tsung Chang, Jaehyeon Park, Wei Zhou, Xiaohe Liu, Barbara Sato, Dominic Dinh, Anna Furimsky, Lucia Beviglia, Lidia Sambucetti, Ling Jong. SRI-28731, a highly potent and selective MAP4K4 (HGK) inhibitor for cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 731. doi:10.1158/1538-7445.AM2014-731

Abstract 4483: SR13668, a selective 12-lipoxygenase inhibitor, suppresses tumor growth by modulating the Akt signaling pathway

SR13668, an orally active cancer therapeutic agent, exhibits potent antitumor activity against various human cancer xenografts (e.g., breast, prostate, lung and ovarian cancer) without significant toxicity. An exploratory IND study of SR13668 has been completed with a two-stage design to compare the bioavailability of five SR13668 formulations, and the preferred formulation will be subjected to investigation in subsequent phase I testing. SR13668 inhibits growth factor-stimulated phosphorylation of Akt and its downstream target GSK3β in PC-3 prostate and high pAkt expressing MDA-MB-468 breast cancer cells in a dose-dependent manner. SR13668 was also shown to block FGF- and IGF-stimulated Akt activation. Screening a broad selection of kinase targets, including Akt(1,2,3), PI3K, and PDK1, indicated that SR13668 is not a kinase inhibitor. Preliminary mechanistic studies showed that SR13668 selectively inhibits 12-lipoxygenase (12-LOX), but does not affect other lipoxygenases (5-LOX and 15-LOX), or cyclooxygenases (COX-1 and COX-2). 12-LOX plays a major role in promoting cancer progression, invasion and metastasis as well as tumor angiogenesis. 12-LOX has been regarded as a novel target for cancer therapy, since it is generally absent in normal epithelia and is often constitutively expressed in various epithelial cancers. Unlike direct inhibitors of PI3K or Akt, SR13668 has no adverse effects on fasting glucose levels or body weights after 14 days of oral treatment with SR13668 at 500 mg/kg/day, a dose more than 10 times higher than that needed for antitumor activity. Pharmacologic intervention studies with lipoxygenase inhibitors for the prevention or inhibition of cancers have been difficult to interpret, as there are very few isoform-selective LOX inhibitors available. SR13668 is a highly specific 12-LOX inhibitor. No off-target activities were observed, when screened against a broad collection of 40 diverse enzymes and 80 transmembrane and soluble receptors, ion channels and monoamine transporters. The Phase 0 clinical study showed that SR13668 has long half-life (11.2 ± 3.1 hours), and patients did not experience significant adverse events related to SR13668 treatment; thus SR13668 is a promising candidate for further clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2011-4483