Cholesterol-induced mammary tumorigenesis is enhanced by adiponectin deficiency: role of LDL receptor upregulation.

Adiponectin is an adipokine that can suppress the proliferation of various human carcinoma cells. Although its anti-tumor activities have been suggested by many clinical investigations and animal studies, the underlying mechanisms are not fully characterized. In MMTV-polyomavirus middle T antigen (MMTV-PyVT) transgenic mice models, reduced- or complete loss-of-adiponectin expression promotes mammary tumor development. The present study demonstrated that while tumor development in control MMTV-PyVT mice is associated with a progressively decreased circulating cholesterol concentration, adiponectin deficient MMTV-PyVT mice showed significantly elevated total- and low density lipoprotein (LDL)-cholesterol levels. Cholesterol contents in tumors derived from adiponectin deficient mice were dramatically augmented. High fat high cholesterol diet further accelerated the tumor development in adiponectin deficient PyVT mice. The protein levels of LDL receptor (LDLR) were found to be upregulated in adiponectin-deficient tumor cells. In human breast carcinoma cells, treatment with LDL-cholesterol or overexpressing LDLR elevates nuclear beta-catenin activity and facilitates tumor cell proliferation. On the other hand, adiponectin decreased LDLR protein expression in breast cancer cells and inhibited LDL-cholesterol-induced tumor cell proliferation. Both in vivo and in vitro evidence demonstrated a stimulatory effect of adiponectin on autophagy process, which mediated the down-regulation of LDLR. Adiponectin-induced reduction of LDLR was blocked by treatment with a specific inhibitor of autophagy, 3-methyladenine. In conclusion, the study demonstrates that adiponectin elicits tumor suppressive effects by modulating cholesterol homeostasis and LDLR expression in breast cancer cells, which is at least in part attributed to its role in promoting autophagic flux.

Adiponectin haploinsufficiency promotes mammary tumor development in MMTV-PyVT mice by modulation of phosphatase and tensin homolog activities.

BACKGROUND: Adiponectin is an adipokine possessing beneficial effects on obesity-related medical complications. A negative association of adiponectin levels with breast cancer development has been demonstrated. However, the precise role of adiponectin deficiency in mammary carcinogenesis remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, MMTV-polyomavirus middle T antigen (MMTV-PyVT) transgenic mice with reduced adiponectin expressions were established and the stromal effects of adiponectin haploinsufficiency on mammary tumor development evaluated. In mice from both FVB/N and C57BL/6J backgrounds, insufficient adiponectin production promoted mammary tumor onset and development. A distinctive basal-like subtype of tumors, with a more aggressive phenotype, was derived from adiponectin haplodeficient MMTV-PyVT mice. Comparing with those from control MMTV-PyVT mice, the isolated mammary tumor cells showed enhanced tumor progression in re-implanted nude mice, accelerated proliferation in primary cultures, and hyperactivated phosphatidylinositol-3-kinase (PI3K)/Akt/beta-catenin signaling, which at least partly attributed to the decreased phosphatase and tensin homolog (PTEN) activities. Further analysis revealed that PTEN was inactivated by a redox-regulated mechanism. Increased association of PTEN-thioredoxin complexes was detected in tumors derived from mice with reduced adiponectin levels. The activities of thioredoxin (Trx1) and thioredoxin reductase (TrxR1) were significantly elevated, whereas treatment with either curcumin, an irreversible inhibitor of TrxR1, or adiponectin largely attenuated their activities and resulted in the re-activation of PTEN in these tumor cells. Moreover, adiponectin could inhibit TrxR1 promoter-mediated transcription and restore the mRNA expressions of TrxR1. CONCLUSION: Adiponectin haploinsufficiency facilitated mammary tumorigenesis by down-regulation of PTEN activity and activation of PI3K/Akt signalling pathway through a mechanism involving Trx1/TrxR1 redox regulations.

The PERK/eIF2 alpha signaling pathway of Unfolded Protein Response is essential for N-(4-hydroxyphenyl)retinamide (4HPR)-induced cytotoxicity in cancer cells.

N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has been tested in clinical trials as a cancer chemopreventive drug. 4HPR is cytotoxic to cancer cells but the underlying molecular mechanisms are at present only partially understood. Here we demonstrate that in the human cervical cancer cell line HeLa and the human leukemia cell line HL-60, 4HPR caused rapid, Reactive Oxygen Species (ROS)-dependent activation of the Unfolded Protein Response (UPR). In HeLa cells, 4HPR was shown to induce cell death and activation of procaspases. These effects of 4HPR could be abolished by the over-expression of dominant negative mutants of PERK or eIF2 alpha. HeLa cells incubated with 4HPR were found to form autophagosomes that were also mediated by the PERK/eIF2 alpha pathway. While 4HPR-induced cell death could be significantly prevented by the presence of specific caspase inhibitors, 3-methyladenine (3-MA) that inhibits autophagosome formation enhanced 4HPR-induced cell death. Examination of individual 4HPR-treated HeLa cells revealed that those without the development of autophagosomes hence exhibiting an incomplete UPR were caspase-active and were not viable, while those with autophagosomes were caspase-inactive and retained cell viability. Our data suggest that the PERK/eIF2 alpha pathway is essential for the cytotoxicity of 4HPR that targets on cancer cells with malfunctional UPR.

ROS mediates 4HPR-induced posttranscriptional expression of the Gadd153 gene.

All-trans-N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that can induce apoptosis in many cancer cell lines. The cytotoxicity of 4HPR is dependent on the production of ROS but the underlying reasons are not entirely certain. We have investigated the role of 4HPR-induced production of ROS in mediating the expression of the recently identified 4HPR-responsive gene Gadd153. In 4HPR-treated cells, the elevation of Gadd153 protein level was prevented by vitamin C, which had no effect on the activation of the Gadd153 gene promoter. The 4HPR-induced elevation of Gadd153 mRNA level persisted even after transcription was blocked with actinomycin D, but declined rapidly upon the addition of antioxidants to the transcription-arrested cells. The mRNA expressed from the full-length Gadd153 cDNA was degraded constitutively in cells in the absence but not in the presence of 4HPR. Such an inhibitory effect of 4HPR was abolished by antioxidants and by inhibitors of 12-lipoxygenase, baicalein (specific) and esculetin (panspecific). The inhibition of 4HPR-induced expression of Gadd153 protein by vitamin C was independent of intracellular proteasome activity and vitamin C had no effect on the intracellular decay of Gadd153 protein. Our data provide the first evidence that the posttranscriptional expression of the Gadd153 gene can be regulated by ROS produced by 4HPR.

The overexpression of Bcl-2 antagonizes the proapoptotic function of the kappa-opioid receptor.

Opioid receptors are G-protein-coupled cell-surface receptors that are mainly expressed in neuronal cells. Stimulation of the kappa-opioid receptor expressed by cultured human epithelial cancer cells promotes staurosporine-induced apoptosis. In this study, while Bcl-2 did not inhibit staurosporine-induced apoptosis, it did inhibit the kappa-opioid receptor-mediated potentiation of apoptosis. The results suggest that Bcl-2 targets a step that is specific to the signaling pathway of the kappa-opioid receptor.

Upregulation of GADD153 expression in the apoptotic signaling of N-(4-hydroxyphenyl)retinamide (4HPR).

The molecular basis for the pharmacologic effects of N-(4-hydroxyphenyl)retinamide (4HPR) was investigated by studying the gene(s) that this compound may upregulate in cultured human epithelial tumor cells. Treatment of the cultured human nasopharyngeal carcinoma-derived cells (CNE3) with 4HPR caused modest cell-cycle arrest at G(1) and apoptosis. The mRNA levels of a total of 20 genes were downregulated with the majority of them involved in cell cycle-related functions. Only the mRNA level of the growth arrest and DNA-damage inducible gene (gadd153) was upregulated by approximately 7-fold, with a concomitant increase in intracellular protein level. Similar upregulation of gadd153 by 4HPR was observed in HeLa and 2 other tumor cell lines. The 4HPR-induced apoptosis was markedly enhanced in the CNE3 cells that transiently overexpressed the gadd153 protein. Unlike 4HPR, all-trans-retinoic acid (ATRA) had no effect on the mRNA or protein level of gadd153. The ability of 4HPR and ATRA to stimulate the promoter activity of gadd153 was then examined. In the HeLa cells, both 4HPR and ATRA caused a 2- to 4-fold stimulation of the promoter activity of gadd153, but similar to the CNE3 cells, ATRA was incapable of upregulating the protein level of gadd153. This is the first demonstration that gadd153 is a 4HPR-responsive gene in tumor cells and may have a functional role to play in 4HPR-induced apoptosis. Furthermore, our data suggest that the expression of gadd153 can be regulated by 4HPR at the transcriptional level.