Irina Budunova

Functional roles of Akt signaling in mouse skin tumorigenesis

The mouse skin carcinogenesis protocol is a unique model for understanding the molecular events leading to oncogenic transformation. Mutations in the Ha-ras gene, and the presence of functional cyclin D1 and the EGF receptor, have proven to be important in this system. However, the signal transduction pathways connecting these elements during mouse skin carcinogenesis are poorly understood. This paper studies the relevance of the Akt and ERK pathways in the different stages of chemically induced mouse skin tumors. Akt activity increases throughout the entire process, and its early activation is detected prior to increased cyclin D1 expression. ERK activity rises only during the later stages of malignant conversion. The observed early increase in Akt activity appears to be due to raised PI-3K activity. Other factors acting on Akt such as ILK activation and decreased PTEN phosphatase activity appear to be involved at the conversion stage. To further confirm the involvement of Akt in this process, PB keratinocytes were transfected with Akt and subsequently injected into nude mice. The expression of Akt accelerates tumorigenesis and contributes to increased malignancy of these keratinocytes as demonstrated by the rate of appearance, the growth and the histological characteristics of the tumors. Collectively, these data provide evidence that Akt activation is one of the key elements during the different steps of mouse skin tumorigenesis.

Increased expression of p50-NF-κB and constitutive activation of NF-κB transcription factors during mouse skin carcinogenesis

To elucidate the possible role of NF-κB in mouse skin carcinogenesis we studied the expression of p50 (NF-κB1), p52 (NF-κB2), p65 (RelA) and IκB-α inhibitor as well as κB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IκB-α protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in κB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive κB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IκB protein Bcl-3 is able to increase p50/p50 homodimer binding to the different κB sites in mouse thymocytes. Our finding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related κB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.

Cell culture assays for chemicals with tumor-promoting or tumor-inhibiting activity based on the modulation of intercellular communication

The ability of chemicals with tumor-promoting or tumor-inhibiting activity to modulate gap junctional intercellular communication is reviewed. The two most extensively used types of assays for screening tests are (1) metabolic cooperation assays involving exchange between cells of precursors of nucleic acid synthesis and (2) dye-transfer assays that measure exchange of fluorescent dye from loaded cells to adjacent cells. About 300 substances of different biological activities have been studied using various assays. For tumor promoters/epigenetic carcinogens, metabolic cooperation assays have a sensitivity of 62% and dye-transfer assays 60%. Thirty percent of DNA-reactive carcinogens also possess the ability to uncouple cells. The complete estimation of the predictive power of these assays could not be made because the majority of the substances studied for intercellular communication effectsin vitro have not yet been studied for promoting activityin vivo. Both metabolic cooperation assays and dye transfer assays respond well to the following classes of substances: phorbol esters, organochlorine pesticides, polybrominated biphenyls, promoters for urinary bladder, some biological toxins, peroxisome proliferators, and some complex mixtures. Results ofin vitro assays for such tumor promoters/nongenotoxic carcinogens, such as some bile acids, some peroxides, alkanes, some hormones, mineral dusts, ascorbic acid, okadaic acid, and benz(e)pyrene, do not correlated with the data ofin vivo two-stage or complete carcinogenesis. Enhancement of intercellular communication was found for 18 chemicals. Among these, cAMP, retinoids, and carotenoids have demonstrated inhibition of carcinogenesis. We examine a number of factors that are important for routine screening, including the requirement for biotransformation for some agents to exert effects on gap junction. We also discuss the mechanisms of tumor promoter and tumor inhibitor effects on gap junctional permeability, including influences of protein kinase activation, changes in proton and Ca2+ intracellular concentrations, and effects of oxy radical production.

Effects of IKK inhibitor PS1145 on NF-κB function, proliferation, apoptosis and invasion activity in prostate carcinoma cells

A key antiapoptotic transcription factor, nuclear factor kappa-B (NF-κB), is known to be critically important for tumor cell growth, angiogenesis and development of metastatic lesions. We and others showed previously that NF-κB transcription factor was constitutively activated in androgen-independent prostate carcinoma (PC) cell lines due to the upregulated activity of inhibitor of NF-κB kinases (IKK). In this work, using luciferase assay, electrophoretic mobility shift assay and Northern blot analysis of expression of endogenous κB-responsive genes, we demonstrate that a novel highly specific small-molecule IKK inhibitor, PS1145, efficiently inhibited both basal and induced NF-κB activity in PC cells. We found that PS1145 induced caspase 3/7-dependent apoptosis in PC cells and significantly sensitized PC cells to apoptosis induced by tumor necrosis factor alpha. We also showed that PS1145 inhibited PC cell proliferation. Effects of PS1145 on proliferation and apoptosis correlated with inhibition of interleukin (IL)-6, cyclin D1, D2, inhibitor of apoptosis (IAP)-1 and IAP-2 gene expression and decreased IL-6 protein level. In addition, we found that incubation with PS1145 inhibited the invasion activity of highly invasive PC3-S cells in invasion chamber assay in a dose-dependent manner. Overall, this study provides the framework for development of a novel therapeutic approach targeting NF-κB transcription factor to treat advanced PC.

Tumor suppressor activity of glucocorticoid receptor in the prostate.

Glucocorticoids are extensively used in combination chemotherapy of advanced prostate cancer (PC). Little is known, however, about the status of the glucocorticoid receptor (GR) in PC. We evaluated over 200 prostate samples and determined that GR expression was strongly decreased or absent in 70–85% of PC. Similar to PC tumors, some PC cell lines, including LNCaP, also lack GR. To understand the role of GR, we reconstituted its expression in LNCaP cells using lentiviral approach. Treatment of LNCaP-GR cells with the glucocorticoids strongly inhibited proliferation in the monolayer cultures and blocked anchorage-independent growth. This was accompanied by upregulation of p21 and p27, down-regulation of cyclin D1 expression and c-Myc phosphorylation. Importantly, the activation of GR resulted in normalized expression of PC markers hepsin, AMACR, and maspin. On the signaling level, GR decreased expression and inhibited activity of the MAP-kinases (MAPKs) including p38, JNK/SAPK, Mek1/2 and Erk1/2. We also found that activation of GR inhibited activity of numerous transcription factors (TF) including AP-1, SRF, NF-κB, p53, ATF-2, CEBPα, Ets-1, Elk-1, STAT1 and others, many of which are regulated via MAPK cascade. The structural analysis of hepsin and AMACR promoters provided the mechanistic rationale for PC marker downregulation by glucocorticoids via inhibition of specific TFs. Our data suggest that GR functions as a tumor suppressor in prostate, and inhibits multiple signaling pathways and transcriptional factors involved in proliferation and transformation.

Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti‐inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable‐sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5‐GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti‐inflammatory role of GR was due at least in part to interference with NF‐κB, leading to a strong reduction in the κB‐binding activity without altering the transcriptional levels of the inhibitor IκBα.

Novel Steroid Receptor Phyto-Modulator Compound A Inhibits Growth and Survival of Prostate Cancer Cells

Androgen receptor (AR)- and glucocorticoid receptor (GR)- mediated signaling play opposite roles in prostate tumorigenesis: AR promotes prostate carcinoma (PC) development, whereas GR acts as a tumor suppressor. Compound A (CpdA) is a stable analogue of an aziridine precursor from the African shrub Salsola tuberculatiformis Botschantzev. It was shown recently that, in model cells, CpdA inhibits AR function and strongly enhances anti-inflammatory function of GR. We determined the effects of CpdA in prostate cells with different AR/GR status: (a) RWPE-1 cells (AR(low)/GR(low)), (b) PC3 and DU145 cells (GR(+)/AR(-)), (c) LNCaP cells (GR(-)/AR(+)), and (d) LNCaP-GR cells expressing both receptors. Similar to steroid hormones, CpdA induces nuclear translocation of both receptors in prostate cells. Despite this, CpdA inhibits DNA-binding and transactivation potential of AR. In addition, CpdA inhibits GR-mediated transactivation but induces GR transrepression via inhibition of several transcription factors, including nuclear factor-kappaB, AP-1, Ets-1, Elk-1, SRF, CRE/ATF, and NFATc. CpdA strongly decreases growth and induces caspase-dependent apoptosis in highly malignant PC3 and DU145 cells and in other AR/GR-expressing PC cells. The cytostatic effect of CpdA is receptor dependent: down-regulation of GR or AR expression drastically attenuates CpdA-induced PC cell growth inhibition. Finally, virtual docking analysis indicates that CpdA shares binding cavities in AR and GR ligand-binding domains with corresponding hormones and forms hydrogen bonds (H-bond) with the same amino acids that are involved in H-bond formation during steroid binding. Overall, our data suggest that CpdA is a unique dual-target steroid receptor modulator that has a high potential for PC therapy.

Androgen receptor targets NFκB and TSP1 to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti‐tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR‐expressing cells formed tumors in male mice at a much lower rate than the AR‐negative controls. Moreover, the AR‐expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti‐angiogenic protein, thrombospondin‐1. AR activation caused a decrease in RelA, a subunit of the pro‐survival transcription factor NFκB, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti‐apoptotic targets, Bcl‐2 and IL‐6. Increased apoptosis within AR‐expressing tumors was likely due to the NFκB suppression, since it was restricted to the cells lacking nuclear (active) NFκB. Thus we for the first time identified combined decrease of NFκB and increased TSP1 as molecular events underlying the AR anti‐tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early‐stage prostate cancer.

Targeting transcription factor NFκB: comparative analysis of proteasome and IKK inhibitors

Nuclear factor-κB (NF-κB) plays a critical role in cancer development and progression. Thus, the NF-κB signaling pathway provides important targets for cancer chemoprevention and anti-cancer chemotherapy. The central steps in NF-κB activation are phosphorylation and proteasome-dependent degradation of its inhibitory proteins termed IκBs. Consequently, the major pharmacological approaches to target NF-κB include (i) repression of IκB kinases (IKKs) and (ii) blocking the degradation of IκBs by proteasome inhibitors. We quantitatively compared the efficacy of various proteasome inhibitors (MG132, lactacystin and epoxomicin) and IKK inhibitors (BAY 11-7082 and PS1145) to block NF-κB activity induced by TNFα or TPA and to sensitize LNCaP prostate carcinoma cells to apoptosis. Our studies revealed significant differences between these two classes of NF-κB inhibitors. We found that proteasome inhibitors epoxomicin and MG132 attenuated NF-κB induction much more effectively than the IKK inhibitors. Furthermore, in contrast to IKK inhibitors, all studied proteasome inhibitors specifically blocked TPA-induced generation de novo of NF-κB p50 homodimers – (p50/p50). These results suggest that the proteasome plays a dominant role in TPA-induced formation of functional p50 homodimers, while IKK activity is less important for this process. Interestingly, profound attenuation of p50/p50 DNA-binding does not reduce the high potency of proteasome inhibitors to suppress NF-κB-dependent transcription. Finally, proteasome inhibitors were much more effective in sensitizing LNCaP cells to TNFα-induced apoptosis compared to IKK inhibitors at the concentrations when both types of agents similarly attenuated NF-κB activity. We conclude that this remarkable pro-apoptotic potential of proteasome inhibitors is partially mediated through NF-κB-independent mechanism.

Differential targeting of androgen and glucocorticoid receptors induces ER stress and apoptosis in prostate cancer cells: A novel therapeutic modality

Androgen (AR) and glucocorticoid (GR) receptor signaling play opposing roles in prostate tumorigenesis: in prostate, AR acts as an oncogene, and GR is a tumor suppressor. Recently, we found that non-steroidal phyto-chemical Compound A (CpdA) is AR/GR modulator acting as anti-inflammatory anti-androgen. CpdA inhibits AR and prevents GR transactivation while enhancing GR transrepression. GR and AR are controlled by proteasomal degradation. We found that prolonged exposure of LNCaP, LNCaP-GR, DU145 and PC3 prostate carcinoma (PCa) cells to proteasome inhibitor Bortezomib (BZ) caused AR degradation and GR accumulation. BZ enhanced CpdA ability to inhibit AR and to augment GR transrepression. We also found that CpdA+BZ differentially regulated GR/AR to cooperatively suppress PCa cell growth and survival and to induce endoplasmic reticulum stress (ERS). Importantly, CpdA+BZ differentially regulated GR-responsive genes. CpdA+BZ blocked activation of glucocorticoid-responsive pro-survival genes, including SGK1, but activated BZ-induced ERS-related genes BIP/HSPA5 and CHOP /GADD153. Using ChIP, we showed that SGK1, BIP/HSPA5 and CHOP regulation was due to effects of CpdA and CpdA+BZ on GR loading on their promoters. We also found that AR and GR are abundant in advanced PCa from patients treated by androgen ablation and/or chemotherapy: 56% of carcinomas from treated patients expressed both receptors, and the other 27% expressed either GR or AR. Overall, our data validate the concept of dual AR/GR targeting in prostate cancer (PC) and suggest that BZ combination with dual-target steroid receptor modulator CpdA has high potential for PC therapy.