Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

The heterozygous R155C VCP mutation: Toxic in humans! Harmless in mice?

Heterozygous missense mutations in the human VCP gene cause inclusion body myopathy associated with Paget disease of bone and fronto-temporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). The exact molecular mechanisms by which VCP mutations cause disease manifestation in different tissues are incompletely understood. In the present study, we report the comprehensive analysis of a newly generated R155C VCP knock-in mouse model, which expresses the ortholog of the second most frequently occurring human pathogenic VCP mutation. Heterozygous R155C VCP knock-in mice showed decreased plasma lactate, serum albumin and total protein concentrations, platelet numbers, and liver to body weight ratios, and increased oxygen consumption and CD8+/Ly6C + T-cell fractions, but none of the typical human IBMPFD or ALS pathologies. Breeding of heterozygous mice did not yield in the generation of homozygous R155C VCP knock-in animals. Immunoblotting showed identical total VCP protein levels in human IBMPFD and murine R155C VCP knock-in tissues as compared to wild-type controls. However, while in human IBMPFD skeletal muscle tissue 70% of the total VCP mRNA was derived from the mutant allele, in R155C VCP knock-in mice only 5% and 7% mutant mRNA were detected in skeletal muscle and brain tissue, respectively. The lack of any obvious IBMPFD or ALS pathology could thus be a consequence of the very low expression of mutant VCP. We conclude that the increased and decreased fractions of the R155C mutant VCP mRNA in man and mice, respectively, are due to missense mutation-induced, divergent alterations in the biological half-life of the human and murine mutant mRNAs. Furthermore, our work suggests that therapy approaches lowering the expression of the mutant VCP mRNA below a critical threshold may ameliorate the intrinsic disease pathology.

Activating ERBB2/HER2 mutations indicate susceptibility to pan-HER inhibitors in Lynch and Lynch-like colorectal cancer.

OBJECTIVE: Microsatellite instability (MSI) is detected in approximately 15% of all colorectal cancers (CRC) and virtually in all cases with Lynch syndrome. The MSI phenotype is caused by dysfunctional mismatch repair (MMR) and leads to accumulation of DNA replication errors. Sporadic MSI CRC often harbours BRAF(V600E); however, no consistent data exist regarding targeted treatment approaches in BRAF(wt) MSI CRC. DESIGN: Mutations and quantitative MSI were analysed by deep sequencing in 196 formalin fixed paraffin embedded (FFPE) specimens comprising Lynch and Lynch-like CRCs from the German Hereditary Nonpolyposis Colorectal Cancer registry. Functional relevance of recurrent ERBB2/HER2 mutations was investigated in CRC cell lines using reversible and irreversible HER-targeting inhibitors, EGFR-directed antibody cetuximab, HER2-directed antibody trastuzumab and siRNA-mediated ERBB2/HER2 knockdown. RESULTS: Quantification of nucleotide loss in non-coding mononucleotide repeats distinguished microsatellite status with very high accuracy (area under curve=0.9998) and demonstrated progressive losses with deeper invasion of MMR-deficient colorectal neoplasms (p=0.008). Characterisation of BRAF(wt) MSI CRC revealed hot-spot mutations in well-known oncogenic drivers, including KRAS (38.7%), PIK3CA (36.5%), and ERBB2 (15.0%). L755S and V842I substitutions in ERBB2 were highly recurrent. Functional analyses in ERBB2-mutated MSI CRC cell lines revealed a differential response to HER-targeting compounds and superiority of irreversible pan-HER inhibitors. CONCLUSIONS: We developed a high-throughput deep sequencing approach for concomitant MSI and mutational analyses in FFPE specimens. We provided novel insights into clinically relevant alterations in MSI CRC and a rationale for targeting ERBB2/HER2 mutations in Lynch and Lynch-like CRC.

NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas.

Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionally, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well.

Blocking the angiopoietin-2-dependent integrin ß-1 signaling axis abrogates small cell lung cancer invasion and metastasis.

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin ß-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin ß-1 or blocking Integrin ß-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin ß-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin ß-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.

EGFR Inhibition Strongly Modulates the Tumour Immune Microenvironment in EGFR-Driven Non-Small-Cell Lung Cancer.

EGFR-driven non-small-cell lung cancer (NSCLC) patients are currently treated with TKIs targeting EGFR, such as erlotinib or osimertinib. Despite a promising initial response to TKI treatment, most patients gain resistance to oncogene-targeted therapy, and tumours progress. With the development of inhibitors against immune checkpoints, such as PD-1, that mediate an immunosuppressive microenvironment, immunotherapy approaches attempt to restore a proinflammatory immune response in tumours. However, this strategy has shown only limited benefits in EGFR-driven NSCLC. Approaches combining EGFR inhibition with immunotherapy to stimulate the immune response and overcome resistance to therapy have been limited due to insufficient understanding about the effect of EGFR-targeting treatment on the immune cells in the TME. Here, we investigate the impact of EGFR inhibition by erlotinib on the TME and its effect on the antitumour response of the immune cell infiltrate. For this purpose, we used a transgenic conditional mouse model to study the immunological profile in EGFR-driven NSCLC tumours. We found that EGFR inhibition mediated a higher infiltration of immune cells and increased local proliferation of T-cells in the tumours. Moreover, inhibiting EGFR signalling led to increased activation of immune cells in the TME. Most strikingly, combined simultaneous blockade of EGFR and anti-PD-1 (aPD-1) enhanced tumour treatment response in a transgenic mouse model of EGFR-driven NSCLC. Thus, our findings show that EGFR inhibition promotes an active and proinflammatory immune cell infiltrate in the TME while improving response to immune checkpoint inhibitors in EGFR-driven NSCLC.

The E3 ligase COP1 promotes ERα signaling and suppresses EMT in breast cancer.

ERα signaling drives proliferation, survival and cancer initiation in the mammary gland. Therefore, it is critical to elucidate mechanisms by which ERα expression is regulated. We show that the tumor suppressor E3 ligase COP1 promotes the degradative polyubiquitination of the microtubule-associated protein HPIP. As such, COP1 negatively regulates estrogen-dependent AKT activation in breast cancer cells. However, COP1 also induces ERα expression and ERα-dependent gene transcription, at least through c-Jun degradation. COP1 and ERα levels are positively correlated in clinical cases of breast cancer. COP1 also supports the metabolic reprogramming by estrogens, including glycolysis. On the other hand, COP1 suppresses EMT in breast cancer cells. COP1 deficiency also contributes to Tamoxifen resistance, at least through protective autophagy. Therefore, COP1 acts as an oncogenic E3 ligase by promoting ERα signaling but also acts as a tumor suppressor candidate by preventing EMT, which reflects a dual role of COP1 in breast cancer.

Notch1 Deficiency Induces Tumor Cell Accumulation Inside the Bronchiolar Lumen and Increases TAZ Expression in an Autochthonous Kras LSL-G12V Driven Lung Cancer Mouse Model.

Purpose: Abrogation of Notch signaling, which is pivotal for lung development and pulmonary epithelial cell fate decisions was shown to be involved in the aggressiveness and the differentiation of lung carcinomas. Additionally, the transcription factors YAP and TAZ which are involved in the Hippo pathway, were recently shown to be tightly linked with Notch signaling and to regulate the cell fate in epidermal stem cells. Thus, we aim to elucidate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression in lung cancer. Methods: We investigated the effect of conditional Cre-recombinase mediated Notch1 knock-out on lung cancer cells in vivo using an autochthonous mouse model of lung adenocarcinomas driven by Kras LSL-G12V and comprehensive immunohistochemical analysis. In addition, we analyzed clinical samples and human lung cancer cell lines for TAZ expression and supported our findings by publicly available data from The Cancer Genome Atlas (TCGA). Results: In mice, we found induction of papillary adenocarcinomas and protrusions of tumor cells from the bronchiolar lining upon Notch1 deficiency. Moreover, the mutated Kras driven lung tumors with deleted Notch1 showed increased TAZ expression and focal nuclear translocation which was frequently observed in human pulmonary adenocarcinomas and squamous cell carcinomas of the lung, but not in small cell lung carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas. Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade.

CD74 is dispensable for development of chronic lymphocytic leukemia in Eµ-TCL1 transgenic mice.

CD74 is a surface protein expressed on immune cells, which acts as receptor for the chemokine macrophage migration inhibitory factor (MIF). Signaling via the MIF/CD74-axis has been reported to be important for the pathogenesis of chronic lymphocytic leukemia (CLL). We wanted to clarify the role of CD74 in MIF-induced signaling/leukemic development. In Eµ-TCL1 transgenic mice, occurrence of the leukemic phenotype was associated with increased surface CD74 expression. Eµ-TCL1+/+Cd74-/- mice showed similar kinetics and clinical features of CLL development as Eµ-TCL1+/+ mice. MIF stimulation of leukemic splenocytes led to AKT activation in a CD74-dependent manner. AKT activation was reduced in Cd74-deficient splenocytes in the presence of the oncogenic TCL1-transgene. Tumor cell apoptosis/proliferation were unaffected in Eµ-TCL1+/+Cd74-/- mice. Our data suggest that the need for active CD74 signaling is overcome in the leukemic context of TCL1-driven CLL, and that CD74 may have a dispensable role for CLL pathogenesis in this model.

The X-linked trichothiodystrophy-causing gene RNF113A links the spliceosome to cell survival upon DNA damage.

Prolonged cell survival occurs through the expression of specific protein isoforms generated by alternate splicing of mRNA precursors in cancer cells. How alternate splicing regulates tumor development and resistance to targeted therapies in cancer remain poorly understood. Here we show that RNF113A, whose loss-of-function causes the X-linked trichothiodystrophy, is overexpressed in lung cancer and protects from Cisplatin-dependent cell death. RNF113A is a RNA-binding protein which regulates the splicing of multiple candidates involved in cell survival. RNF113A deficiency triggers cell death upon DNA damage through multiple mechanisms, including apoptosis via the destabilization of the prosurvival protein MCL-1, ferroptosis due to enhanced SAT1 expression, and increased production of ROS due to altered Noxa1 expression. RNF113A deficiency circumvents the resistance to Cisplatin and to BCL-2 inhibitors through the destabilization of MCL-1, which thus defines spliceosome inhibitors as a therapeutic approach to treat tumors showing acquired resistance to specific drugs due to MCL-1 stabilization.

Inhibition of Tumor VEGFR2 Induces Serine 897 EphA2-Dependent Tumor Cell Invasion and Metastasis in NSCLC.

Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF)-VEGFR2 signaling has shown limited efficacy in lung cancer patients. Here, we demonstrate that inhibition of VEGFR2 in tumor cells, expressed in ∼20% of non-squamous non-small cell lung cancer (NSCLC) patients, leads to a pro-invasive phenotype. Drug-induced inhibition of tumor VEGFR2 interferes with the formation of the EphA2/VEGFR2 heterocomplex, thereby allowing RSK to interact with Serine 897 of EphA2. Inhibition of RSK decreases phosphorylation of Serine 897 EphA2. Selective genetic modeling of Serine 897 of EphA2 or inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, these findings demonstrate that VEGFR2-targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2-targeted anti-angiogenic treatment in lung cancer patients.

Dominant-negative inhibition of Ets 1 suppresses tumor growth, invasion and migration in rat C6 glioma cells and reveals differentially expressed Ets 1 target genes.

We previously reported that the inactivation of the Ets 1 transcription factor by a specific decoy strategy reduces rat C6 glioma cell proliferation and mmp-9 expression. In the present study, we analysed the effects of the dominant-negative form of Ets 1 (Ets-DB) on rat C6 glioma cell proliferation, migration, invasion, in vivo tumor growth on the chicken chorioallantoic membrane (CAM) and mmp-9 expression. In addition, we examined differences in gene expression between Ets-DB expressing and control cells using suppression subtractive hybridization (SSH). We found that retrovirus mediated expression of Ets-DB inhibited cellular proliferation, migration, invasion, mmp-9 expression, cellular growth in soft agar, and in vivo growth in the chicken chorioallantoic membrane assay. SSH analysis revealed expression of different genes in Ets-DB expressing cells involved in basic cellular processes. Each of these genes contained binding sites for different Ets-factors within their promoters. Finally, we found that, in addition to Ets 1, Elk-1, Elf-1, Fli-1 and Etv-1 are further Ets family members expressed in rat C6 glioma cells. Our results indicate that Ets transcription factors play important roles for basic properties of rat C6 glioma cells. Targeting of these factors might therefore become a useful experimental tool for therapeutic strategies against malignant gliomas.

The Endosomal Protein CEMIP Links WNT Signaling to MEK1-ERK1/2 Activation in Selumetinib-Resistant Intestinal Organoids.

MAPK signaling pathways are constitutively active in colon cancer and also promote acquired resistance to MEK1 inhibition. Here, we demonstrate that BRAFV600E -mutated colorectal cancers acquire resistance to MEK1 inhibition by inducing expression of the scaffold protein CEMIP through a ß-catenin- and FRA-1-dependent pathway. CEMIP was found in endosomes and bound MEK1 to sustain ERK1/2 activation in MEK1 inhibitor-resistant BRAFV600E-mutated colorectal cancers. The CEMIP-dependent pathway maintained c-Myc protein levels through ERK1/2 and provided metabolic advantage in resistant cells, potentially by sustaining amino acids synthesis. CEMIP silencing circumvented resistance to MEK1 inhibition, partly, through a decrease of both ERK1/2 signaling and c-Myc. Together, our data identify a cross-talk between Wnt and MAPK signaling cascades, which involves CEMIP. Activation of this pathway promotes survival by potentially regulating levels of specific amino acids via a Myc-associated cascade. Targeting this node may provide a promising avenue for treatment of colon cancers that have acquired resistance to targeted therapies.Significance: MEK1 inhibitor-resistant colorectal cancer relies on the scaffold and endosomal protein CEMIP to maintain ERK1/2 signaling and Myc-driven transcription. Cancer Res; 78(16); 4533-48. ©2018 AACR.

Synergistic anti-angiogenic treatment effects by dual FGFR1 and VEGFR1 inhibition in FGFR1-amplified breast cancer.

FGFR1 amplification has been found in 15% of patients with breast cancer and has been postulated as a promising marker to predict response against FGFR inhibitors. However, early phase clinical trials of selective FGFR inhibitors demonstrated only limited efficacy in FGFR1-amplified breast cancer patients. We found that BGJ398, an FGFR inhibitor, effectively inhibited phosphorylation of FGFR1 and MEK/ERK signaling in FGFR1-amplified breast cancer without affecting tumor cell proliferation. However, FGFR1 knockout inhibited tumor angiogenesis in vivo. We unraveled that FGFR1 regulates the secretion of the proangiogenic vascular endothelial growth factor (VEGF) in a MAPK-dependent manner. We further found that FGF-FGFR1 signaling induces an autocrine activation of VEGF-VEGFR1 pathway that again amplifies VEGF secretion via VEGF-VEGFR1-AKT signaling. Targeting both VEGFR1 and FGFR1 resulted in synergistic anti-angiogenic treatment effects in vivo. We thus postulate synergistic treatment effects in FGFR1/VEGFR1-positive breast cancer patients by dual targeting of FGFR and VEGFR.

LIN28B enhanced tumorigenesis in an autochthonous KRASG12V-driven lung carcinoma mouse model.

LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.

Combined VEGF and PD-L1 Blockade Displays Synergistic Treatment Effects in an Autochthonous Mouse Model of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) represents the most aggressive pulmonary neoplasm and is often diagnosed at late stage with limited survival, despite combined chemotherapies. We show in an autochthonous mouse model of SCLC that combined anti-VEGF/anti-PD-L1-targeted therapy synergistically improves treatment outcome compared with anti-PD-L1 and anti-VEGF monotherapy. Mice treated with anti-PD-L1 alone relapsed after 3 weeks and were associated with a tumor-associated PD-1/TIM-3 double-positive exhausted T-cell phenotype. This exhausted T-cell phenotype upon PD-L1 blockade was abrogated by the addition of anti-VEGF-targeted treatment. We confirmed a similar TIM-3-positive T-cell phenotype in peripheral blood mononuclear cells of patients with SCLC with adaptive resistance to anti-PD-1 treatment. Mechanistically, we show that VEGFA enhances coexpression of the inhibitory receptor TIM-3 on T cells, indicating an immunosuppressive function of VEGF in patients with SCLC during anti-PD-1-targeted treatment. Our data strongly suggest that a combination of anti-VEGF and anti-PD-L1 therapies can be an effective treatment strategy in patients with SCLC.Significance: Combining VEGF and PD-L1 blockade could be of therapeutic benefit to patients with small cell lung cancer. Cancer Res; 78(15); 4270-81. ©2018 AACR.

Ets-1 expression promotes epithelial cell transformation by inducing migration, invasion and anchorage-independent growth.

Ets-1 is the prototype of the family of ETS transcription factors. In human tumors, Ets-1 is expressed in endothelial cells and fibroblasts of the tumor stroma and is proposed to play a role in tumor vascularization and invasion by upregulating expression of matrix-degrading proteases. In human carcinomas, Ets-1 is also expressed by neoplastic cells, but little is known about the functional implications of this observation. We have addressed the role of Ets-1 in epithelial HeLa tumor cells by selecting stably Ets-1 over and underexpressing HeLa cells. Ets-1 expression increases the transformed phenotype of HeLa cells, by promoting cell migration, invasion and anchorage-independent growth, while Ets-1 downregulation reduces cell attachment. In correlation with these results, Ets-1 upregulation increases integrinbeta2 expression but not that of other integrins. These results suggest that, in addition to its role in the tumor stroma, Ets-1 may also promote tumor development and progression by increasing neoplastic transformation.

Tumour-stroma interactions between metastatic prostate cancer cells and fibroblasts.

Previous work has shown the importance of tumour-stroma interactions for prostate cancer development at the primary site. The aim of the present study was to find out whether evidence can be found for a tumour-stroma cross- talk also between metastatic prostate cancer cell lines and non-prostatic stromal fibroblasts which are encountered by metastatic cells at most sites. We addressed this issue in cell culture systems using 3 metastatic human prostate cancer cell lines (LnCaP, PC-3 and DU-145) on the one hand, and a human fibroblast line (HFF, human foreskin fibroblasts) on the other. We incubated fibroblasts with tumour cell- and tumour cells with fibroblast-conditioned media and evaluated several parameters important for the establishment of metastases such as cell proliferation, migration and expression of matrix degrading proteases. We also determined in the conditioned media the concentrations of several growth factors and cytokines which might be responsible for the observed effects. We found that media conditioned by all 3 metastatic prostate cancer cell lines stimulated fibroblast proliferation which corresponds to fibrous stroma induction in vivo. DU-145 cell conditioned media induced in fibroblasts expression of mmp-1 mRNA known to be important for tumour invasion. ELISA assays revealed that tumour cells secrete bFGF, PDGF and TNFalpha known to stimulate fibroblast proliferation and/or MMP-1 expression. Cultivation of DU-145 carcinoma cells in fibroblast conditioned medium resulted in an enhanced proliferation and anchorage-independent growth of this cell line in soft agar. Fibroblast conditioned medium also increased migration of PC-3 cells in the wound assay and slightly augmented mmp-1 expression. KGF (able to stimulate proliferation of normal and neoplastic prostate epithelial cells) was secreted by fibroblasts at higher concentrations than by all 3 tumour cell lines. In addition, fibroblasts secreted TNFalpha, bFGF, PDGF, HGF and also VEGF, the most important factor for tumour vascularization. Our results provide evidence that tumour-stroma interactions do not only exist at the primary site but also between metastatic prostate cancer cell lines and their fibroblastic microenvironment. These interactions, which are mediated through secreted factors, affect several steps of the metastatic cascade including proliferation, anchorage-independent growth, migration and the secretion of matrix-degrading proteases.