Overcoming brain-derived therapeutic resistance in HER2+ breast cancer brain metastasis.

Brain metastasis of HER2+ breast cancer occurs in about 50% of all women with metastatic HER2+ breast cancer and confers poor prognosis for patients. Despite effective HER2-targeted treatments of peripheral HER2+ breast cancer with Trastuzumab +/-HER2 inhibitors, limited brain permeability renders these treatments inefficient for HER2+ breast cancer brain metastasis (BCBM). The scarcity of suitable patient-derived in-vivo models for HER2+ BCBM has compromised the study of molecular mechanisms that promote growth and therapeutic resistance in brain metastasis. We have generated and characterized new HER2+ BCBM cells (BCBM94) isolated from a patient HER2+ brain metastasis. Repeated hematogenic xenografting of BCBM94 consistently generated BCBM in mice. The clinically used receptor tyrosine kinase inhibitor (RTKi) Lapatinib blocked phosphorylation of all ErbB1-4 receptors and induced the intrinsic apoptosis pathway in BCBM94. Neuregulin-1 (NRG1), a ligand for ErbB3 and ErbB4 that is abundantly expressed in the brain, was able to rescue Lapatinib-induced apoptosis and clonogenic ability in BCBM94 and in HER2+ BT474. ErbB3 was essential to mediate the NRG1-induced survival pathway that involved PI3K-AKT signalling and the phosphorylation of BAD at serine 136 to prevent apoptosis. High throughput RTKi screening identified the brain penetrable Poziotinib as highly potent compound to reduce cell viability in HER2+ BCBM in the presence of NRG1. Successful in-vivo ablation of BCBM94- and BT474-derived HER2+ brain tumors was achieved upon two weeks of treatment with Poziotinib. MRI revealed BCBM remission upon poziotinib, but not with Lapatinib treatment. In conclusion, we have established a new patient-derived HER2+ BCBM in-vivo model and identified Poziotinib as highly efficacious RTKi with excellent brain penetrability that abrogated HER2+ BCBM brain tumors in our mouse models.

Human C1q Tumor Necrosis Factor 8 (CTRP8) defines a novel tryptase+ mast cell subpopulation in the prostate cancer microenvironment.

The adipokine C1q Tumor Necrosis Factor 8 (CTRP8) is the least known member of the 15 CTRP proteins and a ligand of the relaxin receptor RXFP1. We previously demonstrated the ability of the CTRP8-RXFP1 interaction to promote motility, matrix invasion, and drug resistance. The lack of specific tools to detect CTRP8 protein severely limits our knowledge on CTRP8 biological functions in normal and tumor tissues. Here, we have generated and characterized the first specific antiserum to human CTRP8 which identified CTRP8 as a novel marker of tryptase+ mast cells (MCT) in normal human tissues and in the prostate cancer (PC) microenvironment. Using human PC tissue microarrays composed of neoplastic and corresponding tumor-adjacent prostate tissues, we have identified a significantly higher number of CTRP8+ MCT in the peritumor versus intratumor compartment of PC tissues of Gleason scores 6 and 7. Higher numbers of CTRP8+ MCT correlated with the clinical parameter of biochemical recurrence. We showed that the human MC line ROSAKIT WT expressed RXFP1 transcripts and responded to CTRP8 treatment with a small but significant increase in cell proliferation. Like the cognate RXFP1 ligand RLN-2 and the small molecule RXFP1 agonist ML-290, CTRP8 reduced degranulation of ROSAKIT WT MC stimulated by the Ca2+-ionophore A14187. In conclusion, this is the first report to identify the RXFP1 agonist CTRP8 as a novel marker of MCT and autocrine/paracrine oncogenic factor within the PC microenvironment.

Novel CTRP8-RXFP1-JAK3-STAT3 axis promotes Cdc42-dependent actin remodeling for enhanced filopodia formation and motility in human glioblastoma cells.

C1q tumor necrosis factor-related peptide 8 (CTRP8) is the least studied member of the C1Q-TNF-related peptide family. We identified CTRP8 as a ligand of the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1) in glioblastoma multiforme (GBM). The CTRP8-RXFP1 ligand-receptor system protects human GBM cells against the DNA-alkylating damage-inducing temozolomide (TMZ), the drug of choice for the treatment of patients with GBM. The DNA protective role of CTRP8 was dependent on a functional RXFP1-STAT3 signaling cascade and targeted the monofunctional glycosylase N-methylpurine DNA glycosylase (MPG) for more efficient base excision repair of TMZ-induced DNA-damaged sites. CTRP8 also improved the survival of GBM cells by upregulating anti-apoptotic BCl-2 and BCL-XL. Here, we have identified Janus-activated kinase 3 (JAK3) as a novel member of a novel CTRP8-RXFP1-JAK3-STAT3 signaling cascade that caused an increase in cellular protein content and activity of the small Rho GTPase Cdc42. This is associated with significant F-actin remodeling and increased GBM motility. Cdc42 was critically important for the upregulation of the actin nucleation complex N-Wiskott-Aldrich syndrome protein/Arp3/4 and actin elongation factor profilin-1. The activation of the RXFP1-JAK3-STAT3-Cdc42 axis by both RXFP1 agonists, CTRP8 and relaxin-2, caused extensive filopodia formation. This coincided with enhanced activity of ezrin, a key factor in tethering F-actin to the plasma membrane, and inhibition of the actin filament severing activity of cofilin. The F-actin remodeling and pro-migratory activities promoted by the novel RXFP1-JAK3-STAT3-Cdc42 axis were blocked by JAK3 inhibitor tofacitinib and STAT3 inhibitor STAT3 inhibitor VI. This provides a new rationale for the design of JAK3 and STAT3 inhibitors with better brain permeability for clinical treatment of the pervasive brain invasiveness of GBM.

Slow Off-Rate Modified Aptamer (SOMAmer) Proteomic Analysis of Patient-Derived Malignant Glioma Identifies Distinct Cellular Proteomes.

Malignant gliomas derive from brain glial cells and represent >75% of primary brain tumors. This includes anaplastic astrocytoma (grade III; AS), the most common and fatal glioblastoma multiforme (grade IV; GBM), and oligodendroglioma (ODG). We have generated patient-derived AS, GBM, and ODG cell models to study disease mechanisms and test patient-centered therapeutic strategies. We have used an aptamer-based high-throughput SOMAscan® 1.3K assay to determine the proteomic profiles of 1307 different analytes. SOMAscan® proteomes of AS and GBM self-organized into closely adjacent proteomes which were clearly distinct from ODG proteomes. GBM self-organized into four proteomic clusters of which SOMAscan® cluster 4 proteome predicted a highly inter-connected proteomic network. Several up- and down-regulated proteins relevant to glioma were successfully validated in GBM cell isolates across different SOMAscan® clusters and in corresponding GBM tissues. Slow off-rate modified aptamer proteomics is an attractive analytical tool for rapid proteomic stratification of different malignant gliomas and identified cluster-specific SOMAscan® signatures and functionalities in patient GBM cells.

Emerging roles for the relaxin/RXFP1 system in cancer therapy.

A role for the hormone relaxin in cancer was described well before the receptor was identified. Relaxin predominantly increases the growth and invasive potential in cancers of different origins. However, relaxin was also shown to promote cell differentiation and to act in a dose-and time-dependent manner in different cancer cell models used. Following the discovery of the relaxin like family peptide receptor 1 (RXFP1) as the cellular receptor for RLN1 and RLN2, research has focussed on the ligand interaction with the large extracellular domain of RXFP1 and resulting molecular signaling mechanisms. RXFP1 activation mediates anti-apoptotic functions, angiogenesis and chemoresistance in cancer cells. This minireview summarizes the known biological functions of RXFP1 activation in different cancer entities in-vitro and in-vivo and outlines possible mechanisms to therapeutically address the relaxin-RXFP1 system in cancer cells.

Inhibitor of DNA Binding 2 Inhibits Epithelial-Mesenchymal Transition via Up-Regulation of Notch3 in Breast Cancer.

Breast cancer is the second leading cause of cancer death in women worldwide. Incurable metastatic breast disease presents a major clinical challenge and is the main cause of breast cancer-related death. The epithelial-mesenchymal transition (EMT) is a critical early promoter of metastasis. In the present study, we identified a novel role for the inhibitor of DNA binding 2 (Id2), a member of the basic helix-loop-helix protein family, during the EMT of breast cancer. Expression of Id2 was positively correlated with Notch3 in breast cancer cells. Low expression of Id2 and Notch3 was associated with worse distant metastasis-free survival in breast cancer patients. The present study revealed that Id2 activated Notch3 expression by blocking E2A binding to an E-box motif in the Notch3 promoter. The Id2-mediated up-regulation of Notch3 expression at both the mRNA and protein levels resulted in an attenuated EMT, which was associated with reduced motility and matrix invasion of ER-positive and -negative human breast cancer cells and the emergence of E-cadherin expression and reduction in the mesenchymal marker vimentin in triple-negative breast cancer cells. In summary, our findings identified Id2 as a suppressor of the EMT and positive transcriptional regulator of Notch3 in breast cancer. Id2 and Notch3 may serve as novel prognostic markers in a subpopulation of ER-positive breast cancer patients.

C1q/TNF-related peptide 8 (CTRP8) promotes temozolomide resistance in human glioblastoma.

The C1q/TNF-related peptide 8 (CTRP8) has recently emerged as a novel ligand of the G protein-coupled receptor RXFP1 in the fatal brain tumor glioblastoma (GBM). We previously demonstrated that the CTRP8-RXFP1 ligand-receptor system promotes motility and matrix invasion of patient GBM and U87 MG cells by specific phosphorylation of PI3 kinase and protein kinase C. Here, we demonstrate a novel role for CTRP8 in protecting human GBM cells against the DNA alkylating damage of temozolomide (TMZ), the standard chemotherapy drug used to treat GBM. This DNA protective role of CTRP8 required a functional RXFP1-STAT3 signaling cascade in GBM cells. We identified N-methylpurine DNA glycosylase (MPG), a monofunctional glycosylase that initiates base excision repair pathway by generating an apurinic/apyrimidinic (AP) site, as a new CTRP8-RXFP1-STAT3 target in GBM. Upon TMZ exposure, treatment with CTRP8 reduced the formation of AP sites and double-strand DNA breaks in GBM cells. This CTRP8 effect was independent of cellular MGMT levels and was associated with decreased caspase 3/7 activity and increased survival of human GBM. CTRP8-induced RXFP1 activation caused an increase in cellular protein levels of the anti-apoptotic Bcl members and STAT3 targets Bcl-2 and Bcl-XL in human GBM. Collectively, our results demonstrate a novel multipronged and clinically relevant mechanism by which the CTRP8-RXFP1 ligand-receptor system exerts a DNA protective function against TMZ chemotherapeutic stress in GBM. This CTRP8-RXFP1-STAT3 axis is a novel determinant of TMZ responsiveness/chemoresistance and an emerging new drug target for improved treatment of human GBM.

Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells.

Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFß1) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFß1 can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFß1 treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFß1 treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFß1-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFß1, in the clinical progression of NSCLC.

Dovitinib enhances temozolomide efficacy in glioblastoma cells.

The multikinase inhibitor and FDA-approved drug dovitinib (Dov) crosses the blood-brain barrier and was recently used as single drug application in clinical trials for GB patients with recurrent disease. The Dov-mediated molecular mechanisms in GB cells are unknown. We used GB patient cells and cell lines to show that Dov downregulated the stem cell protein Lin28 and its target high-mobility group protein A2 (HMGA2). The Dov-induced reduction in pSTAT3Tyr705 phosphorylation demonstrated that Dov negatively affects the STAT3/LIN28/Let-7/HMGA2 regulatory axis in GB cells. Consistent with the known function of LIN28 and HMGA2 in GB self-renewal, Dov reduced GB tumor sphere formation. Dov treatment also caused the downregulation of key base excision repair factors and O6 -methylguanine-DNA-methyltransferase (MGMT), which are known to have important roles in the repair of temozolomide (TMZ)-induced alkylating DNA damage. Combined Dov/TMZ treatment enhanced TMZ-induced DNA damage as quantified by nuclear γH2AX foci and comet assays, and increased GB cell apoptosis. Pretreatment of GB cells with Dov ('Dov priming') prior to TMZ treatment reduced GB cell viability independent of p53 status. Sequential treatment involving 'Dov priming' and alternating treatment cycles with TMZ and Dov substantially reduced long-term GB cell survival in MGMT+ patient GB cells. Our results may have immediate clinical implications to improve TMZ response in patients with LIN28+ /HMGA2+ GB, independent of their MGMT methylation status.

Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells.

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Structural commonality of C1q TNF-related proteins and their potential to activate relaxin/insulin-like family peptide receptor 1 signalling pathways in cancer cells.

We established the role of the GPCR relaxin/insulin-like family peptide receptor 1 (RXFP1 receptor) as a novel active receptor in human glioblastoma (GB), a fatal brain tumour. We identified C1q/TNF-related protein 8 (CTRP8) as a novel agonist of the RXFP1 receptor. CTRP8 enhanced the motility and matrix invasion of GB, and this involved PKC-mediated up-regulation of cathepsin B, a marker for poor prognosis in GB patients. We conclude that the absence of relaxin isoforms does not preclude the activation of the RXFP1 receptor, as the least known member of the CTRP family, CTRP8, can effectively target and activate RXFP1 receptors. LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

RXFP1 is Targeted by Complement C1q Tumor Necrosis Factor-Related Factor 8 in Brain Cancer.

The relaxin-like RXFP1 ligand-receptor system has important functions in tumor growth and tissue invasion. Recently, we have identified the secreted protein, CTRP8, a member of the C1q/tumor necrosis factor-related protein (CTRP) family, as a novel ligand of the relaxin receptor, RXFP1, with functions in brain cancer. Here, we review the role of CTRP members in cancers cells with particular emphasis on CTRP8 in glioblastoma.

Betacellulin transgenic mice develop urothelial hyperplasia and show sex-dependent reduction in urinary major urinary protein content.

The epidermal growth factor (EGF)-like ligands and their cognate ERBB1-4 receptors represent important signaling pathways that regulate tissue and cell proliferation, differentiation and regeneration in a wide variety of tissues, including the urogenital tract. Betacellulin (BTC) can activate all four ERBB tyrosine kinase receptors and is a multifunctional EGF-like ligand with diverse roles in ß cell differentiation, bone maturation, formation of functional epithelial linings and vascular permeability in different organs. Using transgenic BTC mice, we have studied the effect of constitutive systemic BTC over-expression on the urinary bladder. BTC was detected in microvascular structures of the stromal bladder compartment and in umbrella cells representing the protective apical lining of the uroepithelium. ERBB1 and ERBB4 receptors were co-localized in the urothelium. Mice transgenic for BTC and double transgenic for both BTC and the dominant kinase-dead mutant of EGFR (Waved 5) developed hyperplasia of the uroepithelium at 5months of age, suggesting that urothelial hyperplasia was not exclusively dependent on ERBB1/EGFR. Mass spectrometric analysis of urine revealed a significant down-regulation of major urinary proteins in female BTC transgenic mice, suggesting a novel role for systemic BTC in odor-based signaling in female transgenic BTC mice.

C1q-tumour necrosis factor-related protein 8 (CTRP8) is a novel interaction partner of relaxin receptor RXFP1 in human brain cancer cells.

We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain.

Polymeric mesoporous silica nanoparticles as a pH-responsive switch to control doxorubicin intracellular delivery.

Cancer is a leading cause of death. Mesoporous nanomaterials with stimuli sensitivity have received increasing interest as efficient anti-cancer drug carriers. Here, we report hybrid mesoporous nanoparticles of PEGylated silica-poly[2-(dimethylamino)ethyl acrylate] (PEGylated MSN-g-PDMAEA) that can deliver and release the anti-cancer drug doxorubicin (DOX) to tumor cells in a pH dependent switch on/off status. The reversible pH sensitivity resulted in nanoparticles with enhanced functionality through controllable release of the anticancer drug. Confocal laser scanning microscopy (CLSM) and three dimensional (3D) image capture of the intracellular localization of nanoparticles revealed the fast and efficient drug delivery in the human hepatocellular carcinoma cell line. The results highlight the potential of these pH sensitive silica nanoparticles as a novel system for the delivery and controlled release of hydrophobic anti-cancer drugs in the treatment of solid tumors.

Epidermal growth factor cytoplasmic domain affects ErbB protein degradation by the lysosomal and ubiquitin-proteasome pathway in human cancer cells.

The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt), have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22-23-encoded peptide (EGF22.23) enhanced procathepsin B (procathB) expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1). This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

The cytoplasmic domain of proEGF negatively regulates motility and elastinolytic activity in thyroid carcinoma cells.

The intracellular domains of the membrane-anchoring regions of some precursors of epidermal growth factor (EGF) family members have intrinsic biologic activities. We have determined the role of the human proEGF cytoplasmic domain (proEGFcyt) as part of the proEGF transmembrane-anchored region (proEGFctF) in the regulation of motility and elastinolytic invasion in human thyroid cancer cells. We found proEGFctF to act as a negative regulator of motility and elastin matrix penetration and the presence of proEGFcyt or proEGF22.23 resulted in a similar reduction in motility and elastinolytic migration. This activity was counteracted by EGF-induced activation of EGF receptor signaling. Decreased elastinolytic migratory activity in the presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with decreased secretion of elastinolytic procathepsin L. The presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with the specific transcriptional up-regulation of t-SNARE member SNAP25. Treatment with siRNA-SNAP25 resulted in motility and elastin migration being restored to normal levels. Epidermal growth factor treatment down-regulated SNAP25 protein by activating EGF receptor-mediated proteasomal degradation of SNAP25. These data provide first evidence for an important function of the cytoplasmic domain of the human proEGF transmembrane region as a novel suppressor of motility and cathepsin L-mediated elastinolytic invasion in human thyroid carcinoma cells and suggest important clinical implications for EGF-expressing tumors.

Cytoplasmic domain of proEGF affects distribution and post-translational modification of microtubuli and increases microtubule-associated proteins 1b and 2 production in human thyroid carcinoma cells.

We studied the distribution of transcripts encoding the cytoplasmic domain of the membrane-anchored precursor epidermal growth factor (proEGFcyt) and a novel cytoplasmic proEGF splice isoform with a deleted exon 23 and an out-of-frame fusion of exon 24 (proEGFdel23) in human normal and neoplastic thyroid tissues. In papillary thyroid carcinoma (PTC), coexpression of transcripts encoding for both proEGFcyt and proEGFdel23 correlated with poor differentiation of PTC. To determine potential roles of the cytoplasmic proEGF domain in human thyroid cells, we generated stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 overexpressing the normal cytoplasmic domain proEGFcyt, a truncated proEGFcyt composed of the peptide sequence encoded by exons 22 and 23 (proEGF22.23) and proEGFdel23. The proEGFcyt and proEGF22.23 transfectants displayed significantly reduced proliferation rates, an enlarged cellular phenotype, and alterations in the distribution and post-translational modification of the microtubular system. These transfectants also displayed increased production of microtubule-associated proteins 1b and 2c, which was absent in FTC-133-proEGFdel23 or FTC-133-empty plasmid transfectants. This is the first evidence of an involvement of proEGF cytoplasmic domain in microtubular stability in the human thyroid carcinoma cell line FTC-133 and may suggest a specific role for the cytoplasmic domain of membrane-anchored proEGF, particularly exon 23, in thyroid carcinoma. The up-regulation of proEGFdel23 in poorly differentiated PTC and the exclusive detection of both proEGF isoforms in undifferentiated thyroid carcinoma may indicate an involvement of this novel truncated proEGFdel23 cytoplasmic domain during dedifferentiation processes of human thyroid cells.

IGF-I: from diagnostic to triple-helix gene therapy of solid tumors.

Alterations in the expression of growth factors and their receptors are associated with the growth and development of human tumors. One such growth factor is IGF-I (insulin-like growth factor I ), a 70-amino-acid polypeptide expressed in many tissues, including brain. IGF-I is also expressed at high levels in some nervous system-derived tumors, especially in glioblastoma. When using IGF-I as a diagnostic marker, 17 different tumors are considered as expressing the IGF-I gene. Malignant glioma, the most common human brain cancer, is usually fatal. Average survival is less than one year. Our strategy of gene therapy for the treatment of gliomas and other solid tumors is based on: 1) diagnostic using IGF-I gene expression as a differential marker, and 2) application of "triple-helix anti-IGF-I" therapy. In the latter approach, tumor cells are transfected with a vector, which encodes an oligoribonucleotide--an RNA strand containing oligopurine sequence which might be capable of forming a triple helix with an oligopurine and/or oligopyrimidine sequence of the promotor of IGF-I gene (RNA-IGF-I DNA triple helix). Human tumor cells transfected in vitro become down-regulated in the production of IGF-I and present immunogenic (MHC-I and B7 expression) and apoptotic characteristics. Similar results were obtained when IGF-I antisense strategy was applied. In both strategies the transfected cells reimplanted in vivo lose tumorigenicity and elicit tumor specific immunity which leads to elimination of established tumors.