Alexandra M. Fajardo

The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms—induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors—is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

Quantitative and spatial measurements of telomerase reverse transcriptase expression within normal and malignant human breast tissues.

The enzyme telomerase catalyzes the de novo synthesis of telomere repeats, thereby maintaining telomere length, which is necessary for unlimited cellular proliferation. Telomerase reverse transcriptase (TERT), the catalytic domain of telomerase, is the rate-limiting factor for telomerase activity and is expressed in virtually all tumors. Thus, TERT has been proposed as a marker with diagnostic and prognostic potential in breast cancer as well as a basis for breast cancer therapeutics. In these contexts, it is important to define the sites and extent of TERT expression in normal and cancerous human breast tissues. In this study, levels of TERT mRNA were measured within a set of 36 breast carcinomas and 5 normal breast samples by quantitative real-time reverse transcription-PCR, and we subsequently identified and characterized the cells expressing TERT mRNA within these tissues using in situ hybridization. The results show that (a) detectable TERT mRNA expression is specific to the epithelial cells; (b) TERT is expressed in both normal and malignant breast tissues; (c) the pattern and level of TERT expression are heterogeneous, with ∼75% of tumors expressing bulk TERT mRNA levels equal to or less than those within normal breast tissue; and (d) tumors expressing above-normal levels of TERT mRNA are more likely to be histopathologic grade 3 (P = 0.002), contain high fraction of cells in S phase (P = 0.004), and have increased levels of MYC mRNA (P = 0.034).

Chemoprevention in gastrointestinal physiology and disease. Anti-inflammatory approaches for colorectal cancer chemoprevention

Colorectal cancer (CRC) is one of the most common human malignancies and a leading cause of cancer-related deaths in developed countries. Identifying effective preventive strategies aimed at inhibiting the development and progression of CRC is critical for reducing the incidence and mortality of this malignancy. The prevention of carcinogenesis by anti-inflammatory agents including nonsteroidal anti-inflammatory drugs (NSAIDs), selective cyclooxygenase-2 (COX-2) inhibitors, and natural products is an area of considerable interest and research. Numerous anti-inflammatory agents have been identified as potential CRC chemopreventive agents but vary in their mechanism of action. This review will discuss the molecular mechanisms being studied for the CRC chemopreventive activity of NSAIDs (i.e., aspirin, sulindac, and ibuprofen), COX-2 inhibitors (i.e., celecoxib), natural products (i.e., curcumin, resveratrol, EGCG, genistein, and baicalein), and metformin. A deeper understanding of how these anti-inflammatory agents inhibit CRC will provide insight into the development of potentially safer and more effective chemopreventive drugs.

The curcumin analog ca27 down-regulates androgen receptor through an oxidative stress mediated mechanism in human prostate cancer cells

The androgen receptor (AR) plays a critical role in prostate cancer development and progression. Therefore, the inhibition of AR function is an established therapeutic intervention. Since the expression of the AR is retained and often increased in progressive disease, AR protein down‐regulation is a promising therapeutic approach against prostate cancer. We show here that the curcumin analog 27 (ca27) down‐regulates AR expression in several prostate cancer cell lines.

Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel β-carboline derivatives.

By studying the co-crystal information of interactions between PDE5 and its inhibitors, forty new tetrahydro-β-carbolines based-analogues were synthesized, and tested for their PDE5 inhibition. Some compounds were as active as tadalafil in inhibiting PDE5 and of better selectivity profile particularly versus PDE11A, the nature of the terminal ring and its nitrogen substituent are the main determinants of selectivity. Ensemble docking confirmed the role of H-loop closed conformer in activity versus its occluded and open forms. Conformational studies showed the effect of bulkiness of the terminal ring N-alkyl substituent on the formation of stable enzyme ligands conformers. The difference in potencies of hydantoin and piperazinedione analogues, together with the necessity of C-5/C-6 R-absolute configuration has been revealed through molecular docking.

Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells.

Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.

Curcumin Analogs, Oxidative Stress, and Prostate Cancer

Abstract The plant natural product curcumin (diferuloylmethane) and its related naturally occurring curcuminoids are remarkable polyphenols with respect to their many biological activities of interest to human oncology, including antioxidant, anti-inflammatory, antitumorigenic, and antimetastatic effects. Their chemical structures are thus utilized in combinatorial chemistry toward the generation of synthetic analogs in an effort to improve bioavailability and bioefficacy. Traditionally, curcuminoids have been known to be potent antioxidants, neutralizing the deleterious effects of oxidative stress mediated by excess reactive oxygen species, including free radicals. The antioxidant activity constitutes their implication as chemopreventive alimentary components in Southeast Asian populations and their reduced cancer incidence, as well as their use as popular and pharmacologically safe dietary supplements. However, curcuminoids and their analogs have been identified as possible hormetics with respect to their ability to also act as pro-oxidant agents. This chapter focuses on the seemingly paradoxical, dual oxidative effect of curcuminoids as it applies to prostate cancer, the second most lethal malignancy in men in Western countries.

Phosphodiesterase 10A is overexpressed in lung tumor cells and inhibitors selectively suppress growth by blocking β-catenin and MAPK signaling

Phosphodiesterase 10A (PDE10) is a cyclic nucleotide (e.g. cGMP) degrading enzyme highly expressed in the brain striatum where it plays an important role in dopaminergic neurotransmission, but has limited expression and no known physiological function outside the central nervous system. Here we report that PDE10 mRNA and protein levels are strongly elevated in human non-small cell lung cancer cells and lung tumors compared with normal human airway epithelial cells and lung tissue, respectively. Genetic silencing of PDE10 or inhibition by small molecules such as PQ10 was found to selectively inhibit the growth and colony formation of lung tumor cells. PQ10 treatment of lung tumor cells rapidly increased intracellular cGMP levels and activated cGMP-dependent protein kinase (PKG) at concentrations that inhibit lung tumor cell growth. PQ10 also increased the phosphorylation of β-catenin and reduced its levels, which paralleled the suppression of cyclin D1 and survivin but preceded the activation of PARP and caspase cleavage. PQ10 also suppressed RAS-activated RAF/MAPK signaling within the same concentration range and treatment period as required for cGMP elevation and PKG activation. These results show that PDE10 is overexpressed during lung cancer development and essential for lung tumor cell growth in which inhibitors can selectively induce apoptosis by increasing intracellular cGMP levels and activating PKG to suppress oncogenic β-catenin and MAPK signaling.

Abstract 1024: Targeting PAK1 activity in breast cancer: Inhibition of cell growth, survival, motility, and signaling

Breast cancer (BrCa) is one of the most common cancers diagnosed and is the second leading cause of cancer-related deaths of women in the United States. BrCa is divided into four major molecular subtypes; these categories are based upon the expression of hormone receptors (i.e., estrogen receptor (ER) and progesterone receptor (PR)), HER2/neu receptor, and proliferation rate. Of these subtypes triple negative (ER-, PR-, HER2-) BrCa are among the most lethal, are highly aggressive, have poor prognosis, limited treatment options, and lack effective targeted therapies. Our lab has shown p-21 activated kinase 1 (PAK1) protein expression increases with BrCa progression in human tumor samples. PAK1 is highly expressed in invasive tumor tissues in comparison to normal tissues. The role of PAK1 in BrCa development and progression is through its activity as a serine/threonine kinase involved in the regulation of several key oncogenic pathways (i.e., WNT/β-catenin and MAPK/ERK/JNK). Although PAK1 expression is increased in invasive tumor tissues, our studies have found that in human BrCa cell cultures PAK1 expression is independent of hormonal receptor status. Our hypothesis for this study is that triple negative BrCa cell lines are more reliant upon PAK19s activity for cell growth, survival, and motility. To test our hypothesis two triple negative BrCa cell lines, MDA-MB-231 and MDA-MB-468, and two ER+ BrCa cell lines, T47D and ZR75-1, were used. Our results demonstrate there are significantly higher levels of PAK1 protein in T47D and MDA-MB-468, compared to ZR75-1 and MDA-MB-231. To determine if the cells were reliant on the activity of PAK1 for cell viability, a trypan blue exclusion assay was performed. The PAK1 inhibitor 1,1′-Dithiodi-2-napthol (IPA-3) significantly inhibited all four BrCa cell lines, cell growth and viability in a dose-dependent manner. IPA-3 further inhibited cell proliferation of MDA-MB-468 (IC50 = 11.5μM), MDA-MB-231 (IC50 = 14.5μM), ZR75-1 (IC50 = 13.3μM), and T47D (IC50 = 17.1μM). IPA-3 also inhibited the phosphorylation and nuclear translocation of PAK1 in a dose-dependent manner in MDA-MB-231 cells. The inhibition of PAK1 by IPA-3 also significantly inhibited the migration of MDA-MB-231 cells in a time- and dose-dependent manner. The regulation of PAK1 activation by the WNT/β-catenin pathway was further explored in ER+ and triple negative BrCa cell lines. Additional studies are currently underway investigating the role of PAK1 activation on the MAPK/ERK/JNK pathway and its effects on cell migration and invasion. These studies explore the role of PAK-1 in triple negative BrCa cell growth, survival, motility, and oncogenic signaling. Further investigation of PAK19s targets within these signaling pathways will provide opportunities for the development of novel prevention and/or therapeutic strategies. Citation Format: Alexandra M. Fajardo, Tristan Browne, Hannah Graff, Kelly Kleier, Kyle Neltner, Courtney McCall, Brad Meyer, Larry Douglass, Julia Carter. Targeting PAK1 activity in breast cancer: Inhibition of cell growth, survival, motility, and signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1024. doi:10.1158/1538-7445.AM2015-1024

Abstract B39: A novel series of substituted indene derivatives that potently and selectively inhibit growth of tumor cells harboring mutant Ras

Background and Significance: Mutations in the Ras family of proto-oncogenes are relatively common in cancer. Activating Ras mutations occur de novo in approximately one third of cancers and may also develop in tumors that become resistant to targeted therapies, especially against receptor tyrosine kinase inhibitors. The NSAID, sulindac and certain non-cyclooxygenase derivatives are known to have chemopreventive activity and have been reported to be particularly effective against tumors and cell lines with Ras mutations. Although Ras mutations have been known for many years, they remain a target that is poorly represented by current cancer therapeutics. In fact, the target has been described as “undruggable” because of the high affinity of Ras for its substrate, GTP. Methods & Results: Using a synthetic lethal approach, we screened a focused collection of compounds chemically related to sulindac. The screen identified a hit compound that potently and selectively inhibited the growth of tumor cell lines expressing mutant Ras (A-549 and MDA-MB231) compared with a tumor cell line bearing the wild-type Ras (HT-29). Follow-up experiments confirmed that tumor cells of several histotypes expressing mutant Ras were sensitive. The selectivity was also observed in a paired isogenic cell model system in which mutant Ras is artificially introduced (NRK, K-NRK). Based on this scaffold, we have synthesized a series of substituted indene compounds with increased selectivity for cancer cells with mutated Ras. Structure-activity relationships for potency and Ras selectivity are being explored within this series. We have identified highly potent compounds which inhibited growth with IC50 values in the low nM range. The compounds exhibited selectivity ranging between 10- to 500-fold for mutant Ras expressing cell lines. Studies demonstrating the mechanism of growth inhibition as well as studies to identify the molecular target of the prototype compound will be presented. Conclusions: We have synthesized a novel series of compounds that potently inhibit the growth of cancer cell lines of several histotypes harboring Ras mutations. In contrast, tumor cells expressing wild-type Ras are relatively insensitive. This series represents a promising, targeted approach to treatment of cancers with this relatively commonly mutated oncogene. This project is supported by NIH grant 5R01CA155638-02. Citation Format: Xi Chen, Kevin J. Lee, Bernard D. Gary, Joshua C. Canzoneri, Alexandra M. Fajardo, Sara C. Sigler, Veronica Ramirez-Alcantara, Bing Zhu, Gary A. Piazza, Adam B. Keeton. A novel series of substituted indene derivatives that potently and selectively inhibit growth of tumor cells harboring mutant Ras. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B39. doi: 10.1158/1557-3125.RASONC14-B39