Jürgen Dittmer

Interaction of human T-cell lymphotropic virus type I Tax, Ets1, and Sp1 in transactivation of the PTHrP P2 promoter.

We have previously shown that the parathyroid hormone-related protein (PTHrP) promoter contains binding sites for transcription factors Ets1 and Sp1 and that human T-cell lymphotropic virus type I (HTLV-I) Tax cooperates with Ets1 to transactivate the PTHrP P2 promoter. Using the yeast two-hybrid interaction system, we now provide evidence that Tax interacts with Ets1. Moreover, a double mutation (D22A,C23S) in the Tax protein that abrogated the Tax/Ets1 interaction also inhibited the Tax/Ets1 cooperative effect, suggesting that the interaction between Tax and Ets1 is important for transactivation of the PTHrP promoter. In coimmunoprecipitation assays, we find that Tax facilitates the interaction between Ets1 and Sp1, forming a ternary complex. When the Sp1 site in the PTHrP promoter was mutated, the Tax/Ets1 cooperative effect was dramatically decreased. This suggests that Sp1 plays an important role in the Ets1-dependent Tax transactivation of the PTHrP P2 promoter. Finally, we demonstrate that Gal4-Tax is a strong activator of the Gal PTHrP promoter, implying that Tax contributes directly to the transcriptional activation of the promoter. We propose a model in which the Tax/Ets1 cooperative effect on the PTHrP P2 promoter is based on the ability of Tax, Ets1, and Sp1 to form a ternary complex on the template DNA. Tax facilitates the interaction of Ets1/Sp1 and participates directly in the transcription initiation process.

Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells.

The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1–34 and 67–86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin α6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.

Human mesenchymal stem cells induce E-cadherin degradation in breast carcinoma spheroids by activating ADAM10

Mesenchymal stem cells (MSCs) have been shown to communicate with tumor cells. We analyzed the effect of human MSCs (hMSCs) on breast cancer cells in three-dimensional cultures. By using GFP expression and immunohistochemistry, we show that hMSCs invade 3D breast cancer cell aggregates. hMSCs caused breast cancer spheroids to become disorganized which was accompanied by a disruption of cell–cell adhesion, E-cadherin cleavage, and nuclear translocation of E-cadherin, but not by epithelial/mesenchymal transition or by an increase in ERK1/2 activity. In addition, hMSCs enhanced the motility of breast cancer cells. Inhibition of ADAM10 (a disintegrin and metalloprotease 10), known to cleave E-cadherin, prevented both hMSC-mediated E-cadherin cleavage and enhanced migration. Our data suggest that hMSCs interfere with cell–cell adhesion and enhance migration of breast cancer cells by activating ADAM10.

Physical and functional interaction between the human T-cell lymphotropic virus type 1 Tax1 protein and the CCAAT binding protein NF-Y.

Tax1, a potent activator of human T-cell lymphotropic virus type 1 (HTLV-1) transcription, has been shown to modulate expression of many cellular genes. Tax1 does not bind DNA directly but regulates transcription through protein-protein interactions with sequence-specific transcription factors. Using the yeast two-hybrid system to screen for proteins which interact with Tax1, we isolated the B subunit of the CCAAT binding protein NF-Y from a HeLa cDNA library. The interaction of Tax1 with NF-YB was specific in that NF-YB did not interact with a variety of other transcription factors, including human immunodeficiency virus Tat, human papillomavirus E6, and Bicoid, or with the M7 (amino acids 29CP-AS) Tax1 mutant. However, NF-YB did interact with the C-terminal Tax1 mutants M22 (130TL-AS) and M47 (319LL-RS). We also show that in vitro-translated NF-YB specifically bound to a glutathione S-transferase-Tax1 fusion protein. Further, Tax1 coimmunoprecipitated with NF-Y from nuclear extracts of HTLV-1-transformed cells, providing evidence for in vivo interaction of Tax1 and NF-YB. We further demonstrate that Tax1 specifically activated the NF-Y-responsive DQbeta promoter, as well as a minimal promoter which contains only the Y-box element. In addition, mutation of the Y-box element alone abrogated Tax1-mediated activation. Taken together, these data indicate that Tax1 interacts with NF-Y through the B subunit and that this interaction results in activation of the major histocompatibility complex class II promoter. Through activation of this and other NF-Y driven promoters, the Tax1-NF-Y interaction may play a critical role in causing cellular transformation and HTLV-1 pathogenesis.

Mesenchymal stem cells and carcinoma-associated fibroblasts sensitize breast cancer cells in 3D cultures to kinase inhibitors.

Stromal cells, such as mesenchymal stem cells (MSCs) and carcinoma-associated fibroblasts (CAFs), play a role in cancer progression. To analyze their ability to modulate drug response, we generated spheroids of MCF-7 or MDA-MB-231 breast cancer cells in the absence or presence of human (h)MSCs or hCAFs and tested the susceptibility of the breast cancer cells to three different kinase inhibitors (TKI258, RAD001 and RAF265) used in cancer therapy. While stromal cells did not affect the response of either breast cancer cell line to the PDGFR/FGFR/VEGFR inhibitor TKI258, they sensitized breast cancer cells to the mTOR inhibitor RAD001. In MCF-7 cells, this was accompanied by increased apoptosis. hMSCs and to a lesser extent hCAFs also enhanced the cytotoxic effect of RAF inhibitor RAF265 on MDA-MB-231 cells. Searching for the mechanism that underlies the effect of stromal cells on RAF265 response we found that stromal cells inhibited RAF265-induced increase in ERK1/2 phosphorylation, supported RAF265-dependent downregulation of PKCα (protein kinase Cα) and prevented RAF265-induced conversion of LC3B, a marker of autophagy. To mimic the changes in ERK1/2 phosphorylation and PKCα expression in response to the stromal cells, we treated cells with MEK1 inhibitor U0126 or PKCα inhibitor Gö6976, respectively. U0126, but not Gö6976, was as effective as hMSCs in sensitizing MDA-MB-231 cells to RAF265. This suggests that hMSCs and hCAFs increased the cytotoxic effect of RAF265 on MDA-MB-231 cells by downregulating ERK1/2 phosphorylation. In summary, this study shows that hMSCs are able to render breast cancer cells more susceptible to kinase inhibitors and that, to the most part, hCAFs to which hMSCs can differentiate are able to mimic the drug-sensitizing effects of hMSCs.

Ets1 is an effector of protein kinase Calpha in cancer cells

PKCα and Ets1 are both associated with breast cancer progression. Our previous studies suggested that these proteins are likely to functionally interact with one another. Here, we show that attenuation of endogenous PKCα expression (siPα) by RNA interference leads to reduced Ets1 protein expression in a variety of cancer cells. Pulse-chase experiments and treatment with proteasome inhibitor MG-132 revealed that siPα interferes with both Ets1 protein synthesis and stability. The effect of siPα on Ets1 expression could be partially prevented by KN-93, suggesting that calcium/calmodulin-dependent kinase II (CaMKII), a modulator of Ets1 activity, may play a role in PKCα-dependent Ets1 regulation. In contrast, Ets1-regulating kinases ERK1/2 were not found to be involved in this process. To assess the importance of the PKCα/Ets1 interaction, we compared the biological responses of MDA-MB-231 cells to PKCα- and Ets1-specific siRNAs (siE1). While only siPα induced changes in cellular morphology and anchorage-independent growth, both siRNAs similarly affected cellular responses to the antitumor drug mithramycin A and to UV light. Microarray analyses further showed that the expression of a certain set of genes was equally affected by siPα and siE1. The data suggest that Ets1 serves as an effector for PKCα to fulfil certain functions in cancer cells.

Human T-cell Leukemia Virus Type I Tax Protein Transactivates RNA Polymerase III Promoter in Vitro and in Vivo

Tax protein of the human T-cell lymphotropic virus type 1 (HTLV-I) is critical for viral replication and is a potent transcriptional activator of viral and cellular polymerase II (pol II) genes. We report here that Tax is able to transactivate a classical pol III promoter, VA-I. In cotransfection experiments, Tax is shown to increase transcription of the VA-I promoter approximately 25-fold. Moreover, Tax is able to activate VA-I transcription when added exogenously to an in vitro transcription reaction. Using Tax affinity column chromatography, we demonstrate that Tax is able to deplete a HeLa cell extract for components required for transcription of VA-I. The transcriptional activity of the Tax-depleted extract can be restored by the 0.6 M phosphocellulose fraction. Interestingly, a consensus binding site for cAMP-responsive element binding protein (CREB) is located upstream of the VA-I promoter, and deletion of this element results in the loss of Tax responsiveness. When this CREB binding site is replaced by a Gal-4 binding site, the VA-I promoter can be transactivated by a Gal4-Tax fusion protein. Taken together, these results suggest that Tax may activate pol III and pol II promoter through a similar mechanism involving the CREB activation pathway. It is also possible that Tax affects pol III transcription by direct interaction with a component of the pol III transcriptional machinery.

Isolation of a cDNA clone, TRX, encoding a human T-cell lymphotrophic virus type-I Tax1 binding protein

Tax1 is essential for human T-cell lymphotropic virus type I (HTLV-I) virus replication and transformation. We have identified and characterized a Tax1 binding protein, TRX, by cDNA screening of a Jurkat T-cell cDNA library. TRX mRNA is ubiquitously expressed in human tissues tested and cell lines analyzed.

Cyclic AMP Enhances TGFβ Responses of Breast Cancer Cells by Upregulating TGFβ Receptor I Expression

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected.

Stromal cells promote anti-estrogen resistance of breast cancer cells through an insulin-like growth factor binding protein 5 (IGFBP5)/B-cell leukemia/lymphoma 3 (Bcl-3) axis

There is strong evidence that stromal cells promote drug resistance of cancer. Here, we show that mesenchymal stem cells (MSCs) and carcinoma-associated fibroblasts (CAFs) desensitize ERα-positive breast cancer cells to the anti-estrogen fulvestrant. In search for the mechanism, we found that MSCs and CAFs similarly increased the activity of the PI3K/AKT and the JAK/STAT3 pathways and upregulated the expression of integrin β1, IGF1R, HIF1α, CAIX and Bcl-3 in MCF-7 cells. Further analyses revealed that MSCs and CAFs coordinately induce these changes by triggering the downregulation of IGFBP5. Loss of IGFBP5 in MCF-7 cells was an early and long-lasting event in response to MSCs and CAFs and was accompanied by growth stimulation both in the absence and presence of fulvestrant. The growth-stimulatory effect in the absence of fulvestrant could be attributed to PI3K/AKT pathway activation and could be mimicked by insulin. The growth-promoting effect in the presence of fulvestrant depended upon the upregulation of Bcl-3. By cRNA microarray analysis we identified additional IGFBP5 targets, of which two (KLHL4 and SEPP1) were inversely regulated by IGFBP5 and Bcl-3. BT474 cells also responded to stromal cells by downregulating IGFBP5 and upregulating the P-AKT, Bcl-3 and IGF1R levels, whereas T47D cells did not show any of these responses. In conclusion, our data suggest that, by targeting IGFBP5 expression in ERα-positive breast cancer cells, such as MCF-7 cells, MSCs and CAFs are able to orchestrate a variety of events, particularly activation of the PI3K/AKT pathway, upregulation of Bcl-3 expression and desensitization to anti-estrogen.