Peter D. Hobbs

Molecular Determinants of AHPN (CD437)-Induced Growth Arrest and Apoptosis in Human Lung Cancer Cell Lines

ABSTRACT 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor γ-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21 WAF1/CIP1 . In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21 WAF1/CIP1 induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.

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.

A Novel Class of Retinoid Antagonists and Their Mechanism of Action

Retinoids regulate a broad range of biological processes through two subfamilies of nuclear retinoid receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently, we reported a novel type of retinoic acid antagonist (SR11335) and showed that this compound can inhibit retinoic acid (RA)-induced activation of a human immunodeficiency virus type 1 (HIV-1) promoter construct that contains a special RA response element (RARE). We have now further characterized the antagonism mediated by SR11335 and of newly synthesized structurally related compounds. Two compounds, SR11330 and SR11334, which are poor transactivators, also showed antagonist activities, inhibiting all-trans-RA (tRA) and 9-cis-RA. The retinoids inhibited transcriptional activation of RAR/RXR heterodimers effectively, while inhibition of RXR homodimers was less efficient. Inhibition was observed on several RAREs, including the TREpal, βRARE, apoAI-RARE, and CRBPI-RARE. In addition, the antagonists inhibited tRA-induced differentiation of HL-60 cells. The antagonist did not interfere with DNA binding of the receptors. In limited proteolytic digestion assays, SR11335 induced resistance of the receptors to proteolysis, but the pattern of the degradation was not altered from that induced by tRA, suggesting that these antagonists induce their biological effects by competing with agonists for binding to RARs, thereby preventing the induction of conformational changes of the receptors necessary for transcriptional activation.

Aromatic retinoic acid analogs. Synthesis and pharmacological activity

Aromatic analogues of (all-E)- and 13(Z)-retinoic acids have been synthesized as potential chemopreventive agents for the treatment of epithelial cancer. In the E series, (1E,3E)-1-(4-carboxyphenyl)-2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3- butadiene (7a), its ethyl ester 5a, and the epoxy ethyl ester 14 displayed excellent activity in the assay for the inhibition of tumor promotor-induced mouse epidermal ornithine decarboxylase, while (1E,3E)-1-(4-carboethoxy-3-methylphenyl)-2-methyl-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3-butadiene (5b) was inactive. The 13(Z) analogues, (E)-1-(2-carboxyphenyl)-4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3,5-he xatriene (19) and (E)-1-(2-hydroxyphenyl)-4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3,5-he xatriene (27), had minimal activity.

A novel steroidal inhibitor of estrogen-related receptor α (ERRα)

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalphas transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.

Inhibition of mammary carcinoma growth by retinoidal benzoic acid derivatives

The growth of many breast carcinoma cell lines is inhibited by vitamin A, and derivatives as well as synthetic retinoids. New retinoidal derivatives have recently been synthesized. These retinoidal benzoic acid derivatives displayed enhanced potency in their ability to reverse hamster tracheal keratinization and inhibit ornithine decarboxylase induction in mouse epidermis. We therefore screened a series of analogues of these compounds for their ability to inhibit human breast carcinoma cell proliferation utilizing three estrogen receptor-positive and two estrogen receptor-negative cell lines. The compound (E)-4-2-(5,6,7,8)tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)prop enyl benzoic acid (Ro 1374-10) was approximately 2-3 orders of magnitude more potent than all-trans-retinoic acid in inhibiting breast carcinoma cell proliferation while the compound SRI-6409-40, which differs from Ro 1374-10 only by the position of a methyl group, was 50-fold more potent than Ro 1374-10. All of the compounds tested displayed were inactive against the estrogen receptor-negative breast carcinoma lines.

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.

Effects of trans-retinoic acid, 9-cis-retinoic acid, 1α,25-(dihydroxy)vitamin D3 and a novel apoptosis-inducing retinoid on breast cancer and endothelial cell growth

Breast cancer cell growth inhibition was not synergistically enhanced by trans-retinoic acid (RA) or 9-cis-RA plus 1alpha,25-(dihydroxy)vitamin D3 (DHVD). The retinoid/DHVD combinations did lower their 50% effective concentrations for inhibiting retinoid-sensitive MCF-7, but not retinoid-refractory BT-20, breast cancer cell growth. In contrast, the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) and its analog SR11389 inhibited the growth of both cell lines. Unlike RA, 9-cis-RA and DHVD, AHPN and SR11389 also potently inhibited human umbilical vascular endothelial cell growth. These results on AHPN and SR11389 suggest that angiogenesis of tumor microvasculature should also be an effective therapeutic target for this new compound class.

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.