Pasi Pennanen

Antiproliferative Action of Vitamin D

During the past few years, it has become apparent that vitamin D may play an important role in malignant transformation. Epidemiological studies suggest that low vitamin D serum concentration increases especially the risk of hormone-related cancers. Experimentally, vitamin D suppresses the proliferation of normal and malignant cells and induces differentiation and apoptosis. In the present review we discuss the mechanisms whereby vitamin D regulates cell proliferation and whether it could be used in prevention and treatment of hyperproliferative disorders like cancers.

Vitamin D and prostate cancer.

Our recent epidemiological study (Ahonen et al., Cancer Causes Control 11(2000) (847-852)) suggests that vitamin D deficiency may increase the risk of initiation and progression of prostate cancer. The nested case-control study was based on a 13-year follow-up of about 19000 middle-aged men free of clinically verified prostate cancer. More than one-half of the serum samples had 25OH-vitamin D (25-VD) levels below 50 nmol/l, suggesting VD deficiency. Prostate cancer risk was highest among the group of younger men (40-51 years) with low serum 25-VD, whereas low serum 25-VD appeared not to increase the risk of prostate cancer in older men (>51 years). This suggests that VD has a protective role against prostate cancer only before the andropause, when serum androgen concentrations are higher. The lowest 25-VD concentrations in the younger men were associated with more aggressive prostate cancer. Furthermore, the high 25-VD levels delayed the appearance of clinically verified prostate cancer by 1.8 years. Since these results suggest that vitamin D has a protective role against prostate cancer, we tried to determine whether full spectrum lighting (FSL) during working hours could increase serum 25-VD concentrations. After 1-month exposure, there was no significant increase in the serum 25-VD level, although there was a bias towards slightly increasing values in the test group as opposed to decreasing values in controls. There was no significant change in the skin urocanic acid production. The possibility to use FSL in cancer prevention is discussed. In order to clarify the mechanism of VD action on cell proliferation and differentiation, we performed studies with the rat and human prostates as well prostate cancer cell lines. It is possible that 25-VD may have a direct role in the host anticancer defence activity, but the metabolism of vitamin D in the prostate may also play an important role in its action. We raised antibodies against human 1alpha-hydroxylase and 24-hydroxylase. Our preliminary results suggest that vitamin D is actively metabolised in the prostate. Vitamin D appears to upregulate androgen receptor expression, whereas androgens seem to upregulate vitamin D receptor (VDR). This may at least partially explain the androgen dependence of VD action. VD alone or administered with androgen causes a suppression of epithelial cell proliferation. VD can activate mitogen-activated kinases, erk-1 and erk-2, within minutes and p38 within hours. Also, auto/paracrine regulation might be involved, since keratinocyte growth factor (mRNA and protein) was clearly induced by VD. Based on these studies, a putative model for VD action on cell proliferation and differentiation is presented.

The importance of LDL and cholesterol metabolism for prostate epithelial cell growth

Cholesterol-lowering treatment has been suggested to delay progression of prostate cancer by decreasing serum LDL. We studied in vitro the effect of extracellular LDL-cholesterol on the number of prostate epithelial cells and on the expression of key regulators of cholesterol metabolism. Two normal prostatic epithelial cell lines (P96E, P97E), two in vitro immortalized epithelial cell lines (PWR-1E, RWPE-1) and two cancer cell lines (LNCaP and VCaP) were grown in cholesterol-deficient conditions. Cells were treated with 1–50 µg/ml LDL-cholesterol and/or 100 nM simvastatin for seven days. Cell number relative to control was measured with crystal violet staining. Changes in mRNA and protein expression of key effectors in cholesterol metabolism (HMGCR, LDLR, SREBP2 and ABCA1) were measured with RT-PCR and immunoblotting, respectively. LDL increased the relative cell number of prostate cancer cell lines, but reduced the number of normal epithelial cells at high concentrations. Treatment with cholesterol-lowering simvastatin induced up to 90% reduction in relative cell number of normal cell lines but a 15–20% reduction in relative number of cancer cells, an effect accompanied by sharp upregulation of HMGCR and LDLR. These effects were prevented by LDL. Compared to the normal cells, prostate cancer cells showed high expression of cholesterol-producing HMGCR but failed to express the major cholesterol exporter ABCA1. LDL increased relative cell number of cancer cell lines, and these cells were less vulnerable than normal cells to cholesterol-lowering simvastatin treatment. Our study supports the importance of LDL for prostate cancer cells, and suggests that cholesterol metabolism in prostate cancer has been reprogrammed to increased production in order to support rapid cell growth.

Effects of simvastatin, acetylsalicylic acid, and rosiglitazone on proliferation of normal and cancerous prostate epithelial Cells at therapeutic concentrations

Non‐steroidal anti‐inflammatory drugs and cholesterol‐lowering statins have been reported to inhibit prostate cancer cell growth suggesting their chemopreventive potential within the prostate. However, the effect has been demonstrated only with advanced prostate cancer cell lines and with drug concentrations above the clinical therapeutic range. In this study we compared the effect of therapeutic concentrations of acetylsalicylic acid, simvastatin and rosiglitazone on the growth of a set of prostatic primary cultures and various prostate epithelial cell lines.

HDLG5/KIAA0583, encoding a MAGUK‐family protein, is a primary progesterone target gene in breast cancer cells

The steroid hormone progesterone is known to have profound effects on growth and differentiation of normal and malignant breast epithelial cells. The biologic actions of progesterone are exerted through the nuclear progesterone receptor‐mediated control of target gene transcription. We utilized differential display polymerase chain reaction (DD‐RT‐PCR) to identify genes whose expression is altered in response to progestins in cultured breast cancer cells. Here we report identification of a gene encoding a member of the MAGUK protein family, hDlg5 (also known as KIAA0583 and P‐dlg), as being the primary progestin target gene in MCF‐7 breast cancer cells. Quantitative real‐time RT‐PCR analysis showed a rapid and strong upregulation of hDlg5 mRNA in cells treated with synthetic progestin medroxyprogesterone acetate (MPA) in the presence of estrogen in MCF‐7, T47D and ZR‐75‐1 cells. The induction was abrogated by antiprogestin RU486. hDlg5 mRNA was also upregulated by progesterone, R5020 and dexamethasone. Protein synthesis inhibitor cycloheximide failed to block progestin‐mediated induction of the hDlg5 gene. hDlg5 is a member of the growing family of MAGUKs (membrane‐associated guanylate kinase homologs) and is to our knowledge the first member of the family reported to be hormonally regulated. hDlg5 is one of the human homologs of the Drosophila gene dlg [lethal(1)discs‐large], which was initially identified as a tumor suppressor gene. The Dlg has a well‐established role in cell growth control and maintenance of cell adhesion and cell polarity. Domain profile analysis revealed that hDlg5 has 2 additional PDZ domains than previously reported.

Inhibition of FOSL1 overexpression in antiestrogen-resistant MCF-7 cells decreases cell growth and increases vacuolization and cell death

Elevated activator protein-1 (AP-1) activity in breast cancer cells has been linked to Tamoxifen (TAM) resistance. Fos-like antigen-1 (FOSL1) is a member of the AP-1 transcription factor and is overexpressed in a variety of human cancers including breast tumors. We have previously established an estrogen-independent and antiestrogen Toremifene (TOR)-resistant subline of MCF-7 breast cancer cells. In these cells, the expression of FOSL1 is upregulated when compared to the parental cells. In the present study, partial inhibition of FOSL1 expression in these cells by small interfering RNA resulted in a marked decrease of cell growth. The inhibition of cell growth paralleled with changes in cell morphology such as increased formation of vacuoles followed by an increase in the number of dead cells. The inhibition of FOSL1 expression in these cells also restored sensitivity to TOR. Our results suggest that chemotherapy targeting overexpression of FOSL1 could be a potent strategy for treating endocrine resistant breast cancers.

Gene expression changes during the development of estrogen-independent and antiestrogen-resistant growth in breast cancer cell culture models.

We have established estrogen-independent and antiestrogen-resistant cell lines from hormone-dependent MCF-7 breast cancer cells by long-term culture in the absence of estrogen, or in the presence of antiestrogen toremifene, respectively. By using a cDNA microarray we compared gene expression profiles among estrogen-independent, antiestrogen-resistant and long-term estrogen-treated MCF-7 cells. We also determined how the expression of the differentially expressed genes has developed during the long-term culture of the cell lines. Of the screened 1176 cancer-related genes, FOSL1, TIMP1, L1CAM, GDF15, and MYBL2 were found to be differentially expressed between the cell lines. A change in FOSL1 and TIMP1 expression could be attributed to the development of antiestrogen resistance, whereas induced L1CAM expression was implicated in the development of estrogen-independent growth of the cells. Estrogen regulated genes GDF15 and L1CAM became regulated by toremifene in the later passage number of toremifene-resistant cells, which might be an indication of the developed estrogen-agonistic activity of toremifene in these cells. Our findings suggest a pattern where the hormone-responsive cancer cells, which survive E2 deprivation and/or antiestrogen treatment, first acquire necessary changes in gene expression for transition to maximal growth in the new hormonal environment. Then, after prolonged treatment with antiestrogen, the antiestrogen-resistant cells may eventually generate an E2-agonistic response to antiestrogen, probably acquiring additional growth advantage.

Changes in protein tyrosine phosphatase type IVA member 1 and zinc finger protein 36 C3H type-like 1 expression demonstrate altered estrogen and progestin effect in medroxyprogesterone acetate-resistant and estrogen-independent breast cancer cell models

Estrogen stimulates proliferation in hormone-responsive breast cancer cells. Progestins inhibit the estrogen-mediated growth in these cells and are used in the treatment of mammary carcinomas. We applied cDNA microarray and real-time RT-PCR methods to reveal 17beta-estradiol- and medroxyprogesterone acetate (MPA)-regulated genes in MCF-7 breast cancer cells. We identified six genes, two of which were novel MPA and/or 17beta-estradiol-regulated genes: protein tyrosine phosphatase type IVA, member 1 (PTP4A1) and zinc finger protein 36, C3H type-like 1 (ZFP36L1). PTP4A1 expression was upregulated by 17beta-estradiol and this was opposed by MPA treatment of the cells. ZFP36L1 proved to be a direct target of MPA. Since MPA has also been shown to bind to glucocorticoid- and androgen receptors, we studied the regulation of the genes with progesterone, synthetic progestin R5020, dexamethasone and dihydrotestosterone. We also assessed the expression and hormonal regulation of PTP4A1 and ZFP36L1 mRNA in MCF-7-derived MPA-resistant and estrogen-independent sublines. The regulation of PTP4A1 expression upon 17beta-estradiol and combined 17beta-estradiol and MPA treatment was completely reversed in the estrogen-independent and MPA-resistant sublines, respectively. This study suggests an important role for PTP4A1 in cell growth, and shows that MPA may potentiate the effect of 17beta-estradiol in the MPA-resistant breast cancer cells.

The effects of metformin and simvastatin on the growth of LNCaP and RWPE-1 prostate epithelial cell lines.

The anti-diabetic drug metformin and cholesterol-lowering statins inhibit prostate cancer cell growth in vitro and have been linked with lowered risk of prostate cancer in epidemiological studies. We evaluated the effects of these drugs on cancerous and non-cancerous prostate epithelial cell lines. Cancer (LNCaP) and normal (RWPE-1) prostate epithelial cell lines were treated with pharmacologic concentrations of metformin and simvastatin alone and in combinations. Relative changes in cell number were measured with crystal violet staining method. Drug effects on apoptosis and cell cycle were measured with flow cytometry. We also measured changes in the activation and expression of a set of reported target proteins of metformin and statins with Western blotting. Metformin decreased the relative cell number of LNCaP cells by inducing G1 cell cycle block, autophagy and apoptosis, and slightly increased cytosolic ATP levels, whereas RWPE-1 cells were resistant to metformin. However, RWPE-1 cells were sensitive to simvastatin, which induced G2 cell cycle block, autophagy and apoptosis, and increased cytosolic ATP levels in these cells. Combination of metformin and simvastatin synergistically decreased cytosolic ATP levels, increased autophagy and instead of apoptosis, induced necrosis in LNCaP cells. Synergistic effects were not observed in RWPE-1 cells. These results suggest, that prostate cancer cells may be more vulnerable to combined growth-inhibiting effects of metformin and simvastatin compared to normal cells. The data presented here provide evidence for the potency of combined metformin and statin, also at pharmacologic concentrations, as a chemotherapeutic option for prostate cancer.

Abstract A35: LDL induces prostate cancer cell growth and inhibits growth-reducing effects of simvastatin

Background: Several epidemiological studies have reported decreased risk of advanced prostate cancer among men using statins, a widely used group of cholesterol-lowering drugs. The association is possibly linked to serum LDL reduction during statin therapy. We studied in vitro the importance of LDL on growth of normal and cancerous prostate epithelial cells and evaluated its association with growth reducing effect of simvastatin. Methods: Four normal prostatic epithelial cell lines (P96E, P97E, PWR-1E, RWPE-1) and two cancer cell lines (LNCaP and VCaP) were grown in cholesterol-free conditions. Cells were treated with 1 µg/ml, 15 µg/ml or 50 µg/ml LDL, 100 nM or 10 µM simvastatin and/or 100 µM mevalonate for seven days. Cellular growth rate was measured with crystal violet staining. Results: In normal epithelial cells, 1 µg/ml and 15 µg/ml LDL did not have marked effect on cell growth, while 50 µg/ml LDL had a growth reducing effect. Conversely, in cancer cells cellular growth was stimulated in direct correlation with LDL concentration. Simvastatin at 100 nM concentration caused clear growth reduction in normal epithelial cells, and 10 µM simvastatin had a cytotoxic effect. Growth reduction by 100 nM simvastatin was attenuated by increasing LDL concentrations, the effect being completely reversed by 15 µg/ml LDL. However, the cytotoxic effect of 10 µM simvastatin could not be reversed by LDL alone, but required combination of 100 µM mevalonate and 15 µg/ml LDL. Conclusions: Our results demonstrate the importance of LDL for prostate cancer cell growth. The growth reducing effect of simvastatin depends on the level of extracellular LDL. Our study supports the epidemiological studies suggesting that statins’ prostate cancer risk reducing effects depend on LDL reduction. Hypercholesterolemia could be a prostate cancer risk factor. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A35.