Supplementary Figure 2 from Netrin-1 Induces Apoptosis in Human Cervical Tumor Cells via the TAp73α Tumor Suppressor

The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype associated with chemotherapy resistance in epithelial cancers. However, the mechanisms underlying the EMT and its associated signaling dysfunctions are still poorly understood. In two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation, we investigated the status of several signaling elements downstream of G-protein receptors (GPR) and their functional roles in the invasive growth potential. We report that the E-cadherin repressors Slug, Zeb1/2 and Twist are overexpressed in these EMT cells characterized by a triple negative phenotype (loss of estrogen ERα and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the alternative GPR30 estrogen receptor and induction of the invasive growth in collagen type I gels. Ectopic Snail expression suppressed WISP-2 transcripts and down-regulated WISP-2 gene promoter expression in transfected cells. Accordingly, WISP-2 transcripts and Wisp-2 protein were depleted in these two convergent models of BC cell EMT. The EMT caused dominance of several proinvasive pathways downstream of GPR, including GαGβγ subunits, PKCα, AKT and c-Jun induction, constitutive activation of the actin-remodeling GTPase Rac1, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27kip1/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting GαGβγ subunits (BIM-46187, gallein), PKCα (Gö6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin) alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signaling independence to inhibitors of HER family tyrosine kinases and the mitogen- and stress-activated protein kinases. Our study suggests that the signaling protagonists GαGβγ, PKCα and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the management of clinical BC in EMT.

Abnormalities of the p53 tumor suppressor gene are among the most frequent molecular events in human neoplasia. p53 is consequently one of the most studied proteins, and is the subject of over 55,500 scientific papers. In this review, attention is focused on the functions of p53 in DNA repair. We highlight the recent progress in the analysis of protein signals to p53, including PARPs, and ubiquitination cascade proteins MDM2, CRM1, USP10 and 14-3-3σ.

AbstractPyriplatin, cis-diammine(pyridine)chloroplatinum(II), a platinum-based antitumor drug candidate, is a cationic compound with anticancer properties in mice and is a substrate for organic cation transporters that facilitate oxaliplatin uptake. Unlike cisplatin and oxaliplatin, which form DNA cross-links, pyriplatin binds DNA in a monofunctional manner. The antiproliferative effects of pyriplatin, alone and in combination with known anticancer drugs (paclitaxel, gemcitabine, SN38, cisplatin, and 5-fluorouracil), were evaluated in a panel of epithelial cancer cell lines, with direct comparison to cisplatin and oxaliplatin. The effects of pyriplatin on gene expression and platinum–DNA adduct formation were also investigated. Pyriplatin exhibited cytotoxic effects against human cell lines after 24 hours (IC50 = 171–443 μmol/L), with maximum cytotoxicity in HOP-62 non–small cell lung cancer cells after 72 hours (IC50 = 24 μmol/L). Pyriplatin caused a G2-M cell cycle block similar to that induced by cisplatin and oxaliplatin. Induction of apoptotsis and DNA damage response was supported by Annexin-V analysis and detection of phosphorylated Chk2 and H2AX. Treatment with pyriplatin increased CDKN1/p21 and decreased ERCC1 mRNA expression. On a platinum-per-nucleotide basis, pyriplatin–DNA adducts are less cytotoxic than those of cisplatin and oxaliplatin. The mRNA levels of genes implicated in drug transport and DNA damage repair, including GSTP1 and MSH2, correlate with pyriplatin cellular activity in the panel of cell lines. Synergy occurred for combinations of pyriplatin with paclitaxel. Because its spectrum of activity differs significantly from those of cisplatin or oxaliplatin, pyriplatin is a lead compound for developing novel drug candidates with cytotoxicity profiles unlike those of drugs currently in use. Mol Cancer Ther; 10(9); 1709–19. ©2011 AACR.

We have previously shown that the genotoxin-induced apoptosis in mouse embryo fibroblasts was enhanced by the extracellular matrix protein fibronectin (FN). In the present study, we tested the hypothesis that FN regulates the DNA damage response (DDR) signaling pathways in HCT116 (p53-wt) and HT29 (p53-mut) human colon cancer cells and tumor-derived myofibroblasts. DNA damage recognition mechanisms were analyzed by immunofluorescence staining, cell cycle analysis and immunoblotting addressed at specific molecular sensors and executors involved in the DDR pathways. The results show that FN, but not collagen type IV or Matrigel, initiates and potentiates the DDR to the anticancer drug cisplatin in an α5 integrin and cell cycle-dependent manner (S and G2/M phases) in human colon cancer cells. Accordingly, we demonstrate that adhesion of HCT116 cells to FN upregulated the expression of α5 integrin FN receptors at the cell surface. These FN-induced DDR pathways include the concerted phosphorylation of histone H2AX on Ser139 detected as nuclear foci (γ-H2AX, 15 and 25 kDa forms), of ataxia telangiectasia mutated (ATM-Ser1981), checkpoint kinase 2 (CHK2-Thr68, 62 and 67 kDa) and p53-Ser15. These FN-induced γ-H2AX signals were interrupted or attenuated by selective inhibitors acting on the DDR pathway kinases, including wortmannin (targeting the phosphatidylinositol-3-kinase-related protein kinases, PIKK), KU55933 (ATM), NU7026 (DNA-dependent protein kinase catalytic subunit, DNA-PK-cs) and SP600125 (JNK2, stress activated protein kinase/c-Jun N-terminal kinase-2). Adhesion to FN also potentiated the γ-H2AX signals and the cytotoxic effects of cisplatin in human colon tumor-derived myofibroblasts. These data provide evidence that FN promotes DNA damage recognition and chemosensitization to cisplatin via the potentiation of the DNA damage signaling responses in human colon cancer cells and tumor-derived myofibroblasts.

The WNT/β-catenin pathway underlies many human cancers through mutations in the APC, β-catenin, and Axin genes. Activation of WNT signalling can also occur due to the localization of glycogen synthase kinase 3β(GSK3β) to the multivesicular bodies, which prevents the degradation of β-catenin. This leads to accumulation of β-catenin within the cytoplasmic matrix and nucleus of cancer cells, which triggers the transactivation of genes involved in cell proliferation, including various oncogenes. Recent research into the mechanistic regulations of molecule homeostasis and identification of new small-targeted inhibitors has provided further insights into the WNT signalling pathway and its role in human cancers. Novel WNT inhibitors target unsuspected cellular enzymes, such as tankyrases, or casein kinase 1α/γ, which controls the destruction of β-catenin and GSK3β. These could lead to the identification of new biomarkers and WNT-targeted inhibitors for the treatment of cancer.

Identifying molecular factors of sensitivity and resistance of cancer cells to enzastaurin, a drug inhibiting protein kinase C (PKC) beta, remains a major challenge to improve its clinical development. Investigating the cellular effects of enzastaurin in a panel of 20 human cancer cell lines, we found that most cells displaying oncogenic K-Ras mutations also display resistance to enzastaurin. Wild-type (WT) K-Ras cancer cells displaying high sensitivity to enzastaurin also expressed high mRNA levels of epithelial markers, such as E-cadherin (CDH1), and low mRNA expressions of mesenchymal markers, such as vimentin, N-cadherin (CDH2), and other genes frequently expressed in mesenchymal transition such as ZEB1, TWIST, SLUG, SNAIL, and TGFbeta. WT K-Ras enzastaurin-resistant cells also expressed high levels of mesenchymal markers. Based on this observation, the effects of enzastaurin were investigated in epithelial colon COLO205-S cells that expressed WT Ras/Raf and its derived COLO205-R mesenchymal counterpart selected for resistance to most PKC modulators and displaying oncogenic K-Ras (G13D/exon 2). In COLO205-S cells, inhibition of phosphorylated PKCbeta led to the inactivation of AKT and glycogen synthase kinase 3beta and was associated with apoptosis without significant effect on cell cycle progression. In COLO205-R cells, enzastaurin induced mainly necrosis at high concentrations. In COLO205-R cells, a strong activation of extracellular signal-regulated kinase 1/2 possibly due to oncogenic K-Ras was predominantly associated with transcription of potent antiapoptotic genes, such as BCL2, GADD45B, and CDKN1A, as well as the multidrug resistance gene ABCB1. From this study, colon cancer cells undergoing apoptosis under enzastaurin exposure seem to frequently express a WT Ras and an epithelial phenotype.

Tumor invasion is the outcome of a complex interplay between cancer cells and the stromal environment. Considering the contribution of the stromal environment, we developed a membrane-free single-cell and spheroid based complementary model to study cancer invasion through native collagen type-I matrices. Cell morphology is preserved during the assays allowing real time monitoring of invasion-induced changes in cell structure and F-actin organization. Combining these models with computerized quantification permits the calculation of highly reproducible and operator-independent data. These assays are versatile in the use of fluorescent probes and have a flexible kinetic endpoint. Once the optimal experimental conditions are empirically determined, the collagen type-I invasion assays can be used for preclinical validation of small-molecule inhibitors targeting invasion. Initiation and monitoring of the single-cell and spheroid invasion model can be achieved in 8 h (over 3 days) and in 14 h (over 8 days) respectively.

Abstract Background: We evaluated pyriplatin (cis-diammine(pyridine)chloroplatinum(II) or cDPCP), a platinum-based antitumor drug candidate, in vitro using an established cancer cell line panel (Ref. 1) with direct comparison to cisplatin and oxaliplatin. Pyriplatin is a monofunctional, cationic compound with proven anticancer properties in mice (Ref. 2) and recently rediscovered as a substrate for the organic cation transporters, hOCT1 and hOCT2 (Ref. 3). Methods: Antiproliferative effects of pyriplatin given as a single agent for 24 h were evaluated in a panel of 10 human cancer cell lines. Flow cytometry was used to record cell cycle effects after staining cells with propidium iodide and to study apopotosis as imaged with FITC-conjugated annexin V. Effects of combinations of pyriplatin with either taxol, gemcitabine, SN38, cisplatin or 5-fluorouracil were evaluated in parallel with corresponding combinations of either cisplatin or oxaliplatin. Gene expression was determined by quantitative RT-PCR. The formation of platinum-DNA adducts over time was determined by ICP-MS. Results: Pyriplatin exhibited cytotoxic effects against human cell lines after 24 h of treatment (IC50 values: 171 – 443 µM). Pyriplatin continued to be effective over time, with IC50 values decreasing to 24 µM after 72 h in HOP-62 cells, the most sensitive cell line. Antiproliferative effects were also observed at short time points, with IC50 values ranging from 230 to 450 µM at 1, 2 and 5 h. Combinations of pyriplatin with either taxol or cisplatin showed better than additive antiproliferative effects. These results were obtained both for pretreatment of cells with taxol prior to pyriplatin and for simultaneous treatment of cells with cisplatin and pyriplatin. The effects of pyriplatin on cell cycle revealed an accumulation of cells in a sub-G1, or apoptotic phase, after only 24 hours of treatment, which was a result unique from that of either cisplatin or oxaliplatin. Increased apoptosis induction after 24 h relative to cisplatin was confirmed by staining cells with FITC-conjugated Annexin-V. Quantitative RT-PCR results indicated that cells with high levels of RAD50 mRNA are more resistant to pyriplatin, suggesting a role of double-strand breaks and homologous recombination repair in the cellular response to pyriplatin damage. Conclusions: The antiproliferative effects of pyriplatin are unique from those of either cisplatin or oxaliplatin, although the potency of pyriplatin is roughly one order of magnitude lower than either cisplatin or oxaliplatin. Pyriplatin induces apoptosis earlier than either oxaliplatin or cisplatin and the downstream effects of DNA double strand breaks are more evident for pyriplatin than for either oxaliplatin or cisplatin. Support: This work was supported in part by a grant from the U. S. National Cancer Institute. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C100.

Abstract Background: Elisidepsin (PM02734) belongs to Kahalalide F family and displays antiproliferative effects as single agent and in combinations with both cytotoxic and targeted therapies. This study aimed evaluating the role of EMT in resistance to elisidepsin. Materials and Methods: Antiproliferative effects of elisidepsin were evaluated in a panel of colon (HT29, HCT116, COLO205, HCC2998), breast (MCF7, MDA-MB-435, SKBR3), ovarian (OVCAR3, IGROV1), lung (Hop62, Hop92), prostate (PC3, DU145), head&neck (SCC61, SQ20B, HEP2), and pancreatic (MiaPaCa2, CAPAN1) cancer cell lines after 72 hour exposure using MTT assay. Expression of several genes was evaluated using quantitative RT-PCR. Results: Elisidepsin displayed antiproliferative effects against cancer cells with IC50s ranging 0.4’9 µM. As previously reported, cancer cells with high mRNA levels of Erb-B3 also displayed sensitivity to elisidepsin. Consistently, we found that cancer cells displaying an epithelial phenotype, characterized by high levels of mRNA expression of E-cadherin (CDH1), were more sensitive to elisidepsin than mesenchymal cancer cells showing high mRNA and protein levels of vimentin and N-cadherin (CDH2). To further evaluate the role of EMT in resistance to elisidepsin, we studied the antiproliferative effects of elisidepsin in the epithelial colon Colo205-S cells and its derived mesenchymal counterpart Colo205-R cells characterized by the overexpression of the transcription factors SIP1, ZEB1, SNAIL, SLUG, TWIST, and TGFβ. Interestingly, Colo205-S cells were at least 8-fold more sensitive to elisidepsin than Colo205-R cells, IC50s being 0.5 and 4.0µM, respectively. To confirm the role of EMT on resistance to elisidepsin, we used another colon cancer model with inducible SNAIL expression (DLD-1TR21-hSNAIL). In this model, SNAIL induction leads to a mesenchymal phenotype that coincides with loss of aggregation and induction of invasion (EMT). In this model, more than 100-fold SNAIL increase was associated with 12 and 6-fold decrease of E-cadherin and Erb-B3 expression, respectively. Consistent with our hypothesis, we found that the cytotoxicity of elisidepsin was at least 7-fold higher in parental epithelial cells than in SNAIL-induced cells in this model. Conclusions: Sensitivity to elisidepsin correlates with high Erb-B3 and E-cadherin expressions in colon cancer cells. These data suggest that sensitivity to elisidepsin may be dependent of cancer cell differentiation, better results being observed in cancer cells retaining an epithelial phenotype. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B151.

Reduction of epithelial cell–cell adhesion via the transcriptional repression of cadherins in combination with the acquisition of mesenchymal properties are key determinants of epithelial–mesenchymal transition (EMT). EMT is associated with early stages of carcinogenesis, cancer invasion and recurrence. Furthermore, the tumor stroma dictates EMT through intensive bidirectional communication. The pathological analysis of EMT signatures is critically, especially to determine the presence of cancer cells at the resection margins of a tumor. When diffusion barriers disappear, EMT markers may be detected in sera from cancer patients. The detection of EMT signatures is not only important for diagnosis but can also be exploited to enhance classical chemotherapy treatments. In conclusion, further detailed understanding of the contextual cues and molecular mediators that control EMT will be required in order to develop diagnostic tools and small molecule inhibitors with potential clinical implications.

Abstract Netrins and their receptors deleted in colon cancer (DCC), neogenin, UNC5, and integrins are involved in axon guidance, epithelial morphogenesis, vascular pattering, cancer cell survival, invasion, tumor angiogenesis, and metastasis. Here, we considered the possible contribution of the p53-related apoptosis mediators p63 and p73 in the mechanisms underlying the antagonism between netrin-1 and DCC at the cell death control. We have showed that ectopic expression and external addition of netrin-1 in HeLa and HEK-293 cells with inactive p53 lead to impaired cell viability and induction of apoptosis. These responses were associated with up-regulation of the proapoptotic protein TAp73α, decreased Bcl-2/Bax ratio, and caspase-3 cleavage, with no change in protein levels of the antiapoptotic NH2-terminal–truncated ΔNp73α isoform, p73 adapter Yap-1 and p73 E3 ubiquitin ligase Itch, and p63, as well as the transcripts encoding p63, TAp73α, and ΔNp73α. However, the proteasome inhibitor MG132 potentiated, while DCC counteracted, netrin-1–induced TAp73α. Consistently, netrin-1 expression correlated with stabilization of the TAp73α protein and lower levels of TAp73α ubiquitination that was conversely enhanced by DCC, in a netrin-dependent manner. Our data indicate that netrin-1 selectively up-regulates TAp73α by preventing its ubiquitination and degradation. Targeted repression of p73α by shRNA reversed TAp73α and the apoptosis induced by netrin-1, and exacerbated the growth of HeLa tumor xenografts. Apoptosis induced by cisplatin was markedly enhanced in netrin-1 or DCC-expressing cells. Collectively, our data reveal that the transcriptionally active TAp73α tumor suppressor is implicated in the apoptosis induced by netrin-1 in a p53-independent and DCC/ubiquitin-proteasome dependent manner. [Cancer Res 2008;68(20):8231–9]

The mechanisms involved in the epithelial to mesenchymal transition (EMT) are integrated in concert with master developmental and oncogenic pathways regulating in tumor growth, angiogenesis, metastasis, as well as the reprogrammation of specific gene repertoires ascribed to both epithelial and mesenchymal cells. Consequently, it is not unexpected that EMT has profound impacts on the neoplastic progression, patient survival, as well as the resistance of cancers to therapeutics (taxol, vincristine, oxaliplatin, EGF-R targeted therapy and radiotherapy), independent of the "classical" resistance mechanisms linked to genotoxic drugs. New therapeutic combinations using genotoxic agents and/or EMT signaling inhibitors are therefore expected to circumvent the chemotherapeutic resistance of cancers characterized by transient or sustained EMT signatures. Thus, targeting critical orchestrators at the convergence of several EMT pathways, such as the transcription pathways NF-kappa B, AKT/mTOR axis, MAPK, beta-catenin, PKC and the AP-1/SMAD factors provide a realistic strategy to control EMT and the progression of human epithelial cancers. Several inhibitors targeting these signaling platforms are already tested in preclinical and clinical oncology. In addition, upstream EMT signaling pathways induced by receptor and nonreceptor tyrosine kinases (e.g. EGF-R, EGF-R, VEGFR, integrins/FAK, Src) and G-protein-coupled receptors (GPCR) constitute practical options under preclinical research, clinical trials or are currently used in the clinic for cancer treatment: e.g. small molecule inhibitors (Iressa: targeting selectively the EGF-R; CP-751,871, AMG479, NVP-AEW541, BMS-536924, PQIP, AG1024: IGF-R; AZD2171, ZD6474: VEGF-R; AZD0530, BMS-354825, SKI606: Src; BIM-46174: GPCR; rapamycin, CCI-779, RAD-001: mTOR) and humanized function blocking antibodies (Herceptin: ErbB2; Avastin: VEGF-A; Erbitux: EGF-R; Abegrin: alpha nu beta 3 integrins). We can assume that silencing RNA and adenovirus-based gene transfer of therapeutic miR and dominant interferring expression vectors targeting EMT pathways and signaling elements will bring additional ways for the treatment of epithelial cancers. Identification of the factors that initiate, modulate and effectuate EMT signatures and their underlying upstream oncogenic pathways should provide the basis of more efficient strategies to fight cancer progression as well as genetic and epigenetic forms of drug resistance. This goal can be accomplished using global screening of human clinical tumors by EMT-associated cDNA, proteome, miRome, and tissue arrays. (C) 2008 Elsevier Ltd. All rights reserved.

Increased src tyrosine kinase expression and activity has been associated with colon cancer cell invasion and survival. Several signaling pathways are involved in the oncogenic activation of src during the adenoma to carcinoma progression and cellular invasion. In the present study, the synthetic ether lipid analog ET-18-OMe was shown to promote invasion of HCT-8/S11 colon cancer cells into collagen type I through the concomitant activation of src by phosphorylation at Tyr416 (5-30 min) in alpha1-integrin immunoprecipitates containing the integrin binding proteins talin and paxillin, as well as the phoshorylated and activated forms of focal adhesion kinase (FAK) at Tyr397 (a FAK kinase activation signal), Tyr576 and Tyr861. This was associated with the lateral redistribution of alpha1-integrins in focal aggregates and persistent activation of the p130Cas/JNK pathways at 5-30 min, with the subsequent induction and activation of the matrix metalloproteinases MMP-2 and MMP-9 (2-12 h). These activated molecular scaffolds and signaling cascades were not observed in immunoprecipitates of alpha2- and beta1-integrins, and tetraspanin CD9, an invasion and metastasis suppressor linked to integrins and FAK signaling. Our data demonstrate that the lateral redistribution and clustering of alpha1-integrins results in the recruitment of the FAK/src motility-promoting signaling complex involved in cancer cell invasion. Disruption of this proinvasive pathway was accomplished by the dominant negative mutant of src (K295R, kinase dead), src pharmacological inhibitor (PP1) and alpha1-integrin function blocking antibodies. These findings support the notion that the alpha1-integrin- and src-dependent signalosome is a relevant therapeutic target against tumor progression in colon cancer patients.

TFF1 is overexpressed in inflammatory diseases and human cancers of the digestive and urogenital systems. To examine the transforming potential of TFF1 in human colon epithelial cells, premalignant PC/AA/C1 adenoma cells (PC) derived from a patient with familial adenomatous polyposis (FAP) were transformed by the TFF1 cDNA and used as a model of the adenoma–carcinoma transition. Constitutive expression of TFF1 increased anchorage-independent cell growth in soft agar, and induced or potentiated the growth of colon PC-TFF1 and kidney MDCKts.src-TFF1 tumor xenografts in athymic mice. This resulted in reduction of thapsigargin-induced apoptosis and promotion of collagen type I invasion through several oncogenic pathways. Using the differential display approach to identify TFF1 target genes, we found that the dual specific phosphatases Cdc25A and B implicated in cell cycle transitions are strongly upregulated under active forms in both PC-TFF1 and HCT8/S11-TFF1 colon cancer cells. Accordingly, TFF1 expression is absent in normal human colon crypts but is induced in correlation with Cdc25a and b transcript levels and tumor grade in familial and sporadic colon adenomas and carcinomas. We propose that TFF1 and Cdc25A-B cooperate with other dominant oncogenic pathways to induce the adenoma and adenocarcinoma transitions. Agents that target TFF1/Cdc25 signaling pathways may be useful for treating patients with TFF1-positive solid tumors.

We recently showed by DNA microarray analysis that vascular endothelial growth factor (VEGF) receptor (VEGFR) is expressed in HCT8/S11 human colon cancer cells, suggesting that several angiogenic factors may target colon cancer cells themselves. In this study, transcripts encoding the VEGF-165 and semaphorin 3A (Sema3A) receptors and coreceptors Flt-1, KDR/Flk-1, plexin A1, and neuropilins NP-1 and NP-2 were identified by reverse transcription-PCR in the human colon cancer cell lines HCT8/S11, HT29, HCT116, and PCmsrc. Collagen invasion induced by VEGF-165 and Sema3A in HCT8/S11 cells (EC50, 0.4-1 nmol/L) required p42/44 mitogen-activated protein kinase and signaling through RhoA/Rho-kinase-dependent and -independent pathways, respectively. As expected, the VEGFR signaling inhibitor ZD4190 selectively abrogated the proinvasive activity of VEGF in collagen gels (IC50, 10 nmol/L) and chick heart fragments. We identify a novel function for VEGF-165 and Sema3A as proinvasive factors for human colorectal cancer cells. Interestingly, oral administration of the single drug ZD4190 to athymic mice (50 mg/kg/d, once daily) inhibited by 70% the growth of HCT8/S11 tumor cell xenografts. Combinations between the src tyrosine kinase inhibitor M475271 and ZD4190 or cisplatin resulted in additive therapeutic activity against LNM35 human lung tumor xenografts. Our data have significant implications for new therapeutic approaches and individualized treatment targeting VEGFR and src signaling pathways in combination with established clinical drugs at primary tumors and distant metastases in colon and lung cancer patients.