Molecular characterization of acquired tolerance of tumor cells to picropodophyllin (PPP).

BACKGROUND: Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. METHODOLOGY/PRINCIPAL FINDINGS: Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. CONCLUSIONS: Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.

Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9.

The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclear IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the ß-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7- higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.

Aberrant intracellular IGF-1R beta-subunit makes receptor knockout cells (IGF1R-/-) susceptible to oncogenic transformation.

Insulin-like growth factor 1 receptor (IGF-1R) is important for transformation of cells with cellular and viral oncogenes. This knowledge is mainly based on experiments on IGF-1R knockout mouse fibroblasts, which mostly are unable to transform after introduction of various oncogenes. Recently, we observed two variants of R- cells, one of which (R-s) surprisingly expresses the beta-subunit of IGF-1R whereas the other one (R-r) does not. Here we show that the beta-subunit is localized intracellularly and forms perinuclear aggregates. It expresses tyrosine kinase activity and appears to be crucial for cell survival since knockdown of it kills the R-s cells. H-RasV12 and/or polyoma middle T-antigen fail to transform R-r, whereas R- cells expressing the beta-subunit were transformed as assessed by formation of colonies in soft agar. The oncogenic transformation of R-s cells was, however, abrogated when the aberrant beta-subunit was knockdown by siRNA. The occurrence of intracellular IGF-1R, especially in tumor cells, has been widely reported but its function has not been understood. Our study provides evidence that it may be important for cell survival and transformation.

Inhibition of VEGF secretion and experimental choroidal neovascularization by picropodophyllin (PPP), an inhibitor of the insulin-like growth factor-1 receptor.

INTRODUCTION: Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration (AMD) and a leading cause of vision loss. Along with other angiogenic factors like vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1) and its receptor, IGF-1R, have been implicated in CNV. PURPOSE: We have previously shown that the cyclolignan picropodophyllin (PPP) efficiently blocks the insulin-like growth factor-1 receptor (IGF-1R) activity and causes cell death in uveal melanoma cell lines and in an in-vivo model. In this study we investigated the effect of PPP on VEGF expression both in vitro and in vivo and whether this effect has anti-angiogenic consequences in a murine CNV model. MATERIALS AND METHODS: C57BL/6J mice with laser-induced CNVs were treated with PPP. Effects on CNV area were assayed by image analysis. VEGF levels in choroids and retinal pigment epithelial cells (APRE-19) were measured by Western blot or ELISA. Transcriptional activation of the VEGF promoter was determined by luciferase reporter gene assay. RESULTS: Mice treated with PPP, administered intraperitoneally or orally, showed 22-32% (p = 0.002) decrease in CNV area. Furthermore, VEGF levels in the choroids were significantly reduced. In cultured APRE-19 cells, IGF-1 was shown to increase VEGF secretion. This increase was completely blocked by PPP. We could confirm that PPP reduced the level of transcriptional activity of VEGF promoter. CONCLUSIONS: PPP reduces IGF-1 dependent VEGF expression and CNV in vivo. Accordingly, IGF-1R inhibitors may be useful tools in the therapy of conditions associated with CNV including neovascular AMD.

Inhibition of VEGF secretion and experimental choroidal neovascularization by picropodophyllin (PPP), an inhibitor of the insulin-like growth factor-1 receptor.

INTRODUCTION: Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration (AMD) and a leading cause of vision loss. Along with other angiogenic factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and its receptor, IGF-1R, have been implicated in CNV. PURPOSE: A prior study has shown that the cyclolignan picropodophyllin (PPP) efficiently blocks the insulin-like growth factor-1 receptor (IGF-1R) activity and causes cell death in uveal melanoma cell lines and in an in vivo model. In this study we investigated the effect of PPP on VEGF expression, both in vitro and in vivo, and whether this effect has antiangiogenic consequences in a murine CNV model. METHODS: C57BL/6J mice with laser-induced CNVs were treated with PPP. Effects on CNV area were assayed by image analysis. VEGF levels in the choroid and retinal pigment epithelial cells (ARPE-19) were measured by Western blot or ELISA. Transcriptional activation of the VEGF promoter was determined by luciferase reporter gene assay. RESULTS: Mice treated with PPP, administered intraperitoneally or orally, showed a 22% to 32% (P = 0.002) decrease in CNV area. Furthermore, VEGF levels in the choroid were significantly reduced. In cultured ARPE-19 cells, IGF-1 was shown to increase VEGF secretion. This increase was completely blocked by PPP. PPP reduced the level of transcriptional activity of the VEGF promoter. CONCLUSIONS: PPP reduces IGF-1-dependent VEGF expression and CNV in vivo. Accordingly, IGF-1R inhibitors may be useful tools in the treatment of conditions associated with CNV, including neovascular AMD.

Insulin-like growth factor type-I receptor-dependent phosphorylation of extracellular signal-regulated kinase 1/2 but not Akt (protein kinase B) can be induced by picropodophyllin.

The initial event upon binding of insulin-like growth factor 1 to the insulin-like growth factor type-I receptor (IGF-1R) is auto-phosphorylation of tyrosine residues within the activation loop of the kinase domain followed by phosphorylation of other receptor tyrosine residues and the subsequent activation of the intracellular signaling cascades. We found recently that the cyclolignan picropodophyllin (PPP) inhibits phosphorylation of IGF-1R and phosphatidyl-3 kinase/Akt (protein kinase B) signaling molecules without interfering with the highly homologous insulin receptor. Furthermore, PPP causes regression of tumor grafts and substantially prolongs the survival of animals with systemic tumor disease. It is of interest that we show here that short treatments with PPP activate the intracellular extracellular signal-regulated kinase (ERK) signaling. Our data suggest that PPP induces IGF-1R ubiquitination and in turn activates ERK1/2. The PPP-induced ERK activation requires IGF-1R because PPP is not able to induce ERK phosphorylation in IGF-1R-negative cells or in cells in which the receptor is knocked down by small interfering RNA. Moreover, in the absence of Mdm2, an E3 ligase that has been shown previously to be involved in IGF-1R ubiquitination, the phosphorylation of ERK did not occur. Thus, apart from inhibiting the receptor activity, PPP can induce IGF-1R ubiquitination and stimulate ERK in an Mdm2-dependent manner. This response could contribute to the apoptotic effect of PPP.

Beta-arrestin and Mdm2 mediate IGF-1 receptor-stimulated ERK activation and cell cycle progression.

Beta-arrestin1, which regulates many aspects of seven transmembrane receptor (7TMR) biology, has also been shown to serve as an adaptor, which brings Mdm2, an E3 ubiquitin ligase to the insulin-like growth factor-1 receptor (IGF-1R), leading to its proteasome-dependent destruction. Here we demonstrate that IGF-1R stimulation also leads to ubiquitination of beta-arrestin1, which regulates vesicular trafficking and activation of ERK1/2. This beta-arrestin1-dependent ERK activity can occur even when the classical tyrosine kinase signaling is impaired. siRNA-mediated suppression of beta-arrestin1 in human melanoma cells ablates IGF-1-stimulated ERK and prolongs the G1 phase of the cell cycle. These data suggest that beta-arrestin-dependent ERK signaling by the IGF-1R regulates cell cycle progression and may thus be an important regulator of the growth of normal and malignant cells.

IGF-1R tyrosine kinase expression and dependency in clones of IGF-1R knockout cells (R-).

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R-) represent a useful tool for molecular mapping of biological properties of the receptor. R- cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R- cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R- (R-s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R-s revealed expression of a 90 kDa protein being reactive to IGF-1R beta-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R-s survival. Taken together, our study suggests that clones of R- express IGF-1R activity and dependency, which in turn may explain that R- can undergo spontaneous transformation.

Expression and growth dependency of the insulin-like growth factor I receptor in craniopharyngioma cells: a novel therapeutic approach.

Craniopharyngioma is a rare benign intracranial epithelial tumor that, however, often recurs and sometimes kills the affected patients, one-third of which are children. In many cases, the patients acquire growth hormone deficiency and postoperatively need substitution. Generally, growth hormone promotes local release of insulin-like growth factor I (IGF-I), which in turn activates the IGF-I receptor (IGF-IR) if present. Together, these circumstances raise the question whether IGF-IR may be involved in craniopharyngioma growth. To address this issue, we analyzed phenotypically well-characterized primary low-passage craniopharyngioma cell lines from nine different patients for IGF-IR expression and IGF-I dependency. Two of the cell lines showed no/very low expression of the receptor and was independent on IGF-I, whereas five cell lines exhibited a strong expression and was clearly contingent on IGF-I. The two remaining cell lines had low receptor expression and IGF-I dependency. Upon treatment with an IGF-IR inhibitor, cells with high IGF-IR expression responded promptly with decreased Akt phosphorylation followed by growth arrest. These responses were not seen in cells with no/very low receptor expression. Growth of cell lines with low IGF-IR expression was only slightly affected by IGF-IR inhibition. Taken together, our data suggest that IGF-IR may be involved in the growth of a subset of craniopharyngiomas and points to the possibility of the involvement of IGF-IR inhibitors as a treatment modality to obtain complete tumor-free conditions before growth hormone substitution.

The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway.

The insulin-like growth factor-1 receptor (IGF-1R) is crucial for many functions in neoplastic cells, for example, antiapoptosis. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. PPP preferentially reduced phosphorylated Akt, as compared to phosphorylated Erk1/2, and caused apoptosis. Now, we aimed to investigate how PPP inhibits the IGF-1R tyrosine kinase (IGF-1RTK) and the PI3K/Akt apoptotic pathway. Using a baculovirus driven IGF-1RTK we found that PPP interfered with tyrosine phosphorylation in the activation loop of the kinase domain. Specifically, it blocked phosphorylation of tyrosine (Y) 1136, while sparing the two others (Y1131 and Y1135). To explore the impact of inhibition of Y1136 on Akt phosphorylation we transfected P6 cells (overexpressing IGF-1R) and malignant melanoma cells with different IGF-1R mutants, including Y1136F (tyrosine replaced by phenylalanine). Y1136F was found to strongly decrease IGF-1 stimulated phosphorylation of Akt. Conversely, Akt phosphorylation was weakly affected in the Y1131F transfectant. Taken together, our data suggest that the preferential inhibition of phosphorylated Akt, after PPP treatment, may be due to specific inhibition of Y1136. PPP was proven not to interfere directly with Akt or any of its downstream molecules in the apoptotic pathway.