Enhances Tumor Cell Motility Research Articles

Semaphorin 4D, a lymphocyte semaphorin, enhances tumor cell motility through binding its receptor, plexinB1, in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor, for which the development of new biomarkers and therapeutic targets has become critical. The main cause of poor prognosis in PDAC patients is the high invasive and metastatic potential of the cancer. In the present study, we report a new signaling pathway that was found to mediate the enhanced tumor cell motility in pancreatic cancer. Semaphorin 4D (Sema4D) is a ligand known to be expressed on different cell types, and has been reported to be involved in the regulation of immune functions, epithelial morphogenesis, and tumor growth and metastasis. In this study, we revealed for the first time that the cancer tissue cells expressing Sema4D in PDAC are tumor-infiltrating lymphocytes. The overexpression of Sema4D and of its receptor, plexinB1, was found to be significantly correlated with clinical factors, such as lymph node metastasis, distant metastasis, and poor prognosis in patients with PDAC. Through in vitro analysis, we demonstrated that Sema4D can potentiate the invasiveness of pancreatic cancer cells and we identified the downstream molecules. The binding of Sema4D to plexinB1 induced small GTPase Ras homolog gene family, member A activation and resulted in the phosphorylation of MAPK and Akt. In addition, in terms of potential therapeutic application, we clearly demonstrated that the enhanced-cell invasiveness induced by Sema4D could be inhibited by knockdown of plexinB1, suggesting that blockade of plexinB1 might diminish the invasive potential of pancreatic cancer cells. Our findings provide new insight into possible prognostic biomarkers and therapeutic targets in PDAC patients.

Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release

In solid malignant tumors, lactate has been identified as a prognostic parameter for metastasis and overall survival of patients. To investigate the effects of lactate on tumor cell migration, Boyden chamber assays were applied. We could show here that lactate enhances tumor cell motility of head and neck carcinoma cell lines significantly in a dose-dependent manner. The changes in tumor cell migration could be attributed to L-lactate or a conversion of lactate to pyruvate, as only these two substances were able to increase migration. Addition of D-lactate or changes in osmolarity or intracellular pH did not alter the migratory potential of the cells investigated. Because lactate was shown earlier to impair the penetration of dendritic cells in a tumor spheroid model, which is contrary to the response of the malignant cell population in the present study, we included blood monocytes in our assay as a highly motile immune cell type and precursor of tumor-associated macrophages. Interestingly, high levels of L-lactate (20 mM) at a pH of 7.4 inhibited monocyte migration in the Boyden chamber system. In addition, cytokine release of TNF and IL-6 was inhibited. The obtained data suggest that high lactate content promotes tumor progression by contributing to the phenomenon of tumor immune escape and by enhancing the migratory potential of the malignant cell population which may directly be coupled to a higher incidence of metastasis.

IL-6 Stabilizes Twist and Enhances Tumor Cell Motility in Head and Neck Cancer Cells through Activation of Casein Kinase 2

BackgroundSquamous cell carcinoma of the head and neck (SCCHN) is the seventh most common cancer worldwide. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years, and thus new targets for therapy are needed. Recently, elevations in serum level of interleukin 6 (IL-6) and expression of Twist in tumor samples were found to be associated with poor clinical outcomes in multiple types of cancer, including SCCHN. Although Twist has been proposed as a master regulator of epithelial-mesenchymal transition and metastasis in cancers, the mechanisms by which Twist levels are regulated post-translationally are not completely understood. Tumor progression is characterized by the involvement of cytokines and growth factors and Twist induction has been connected with a number of these signaling pathways including IL-6. Since many of the effects of IL-6 are mediated through activation of protein phosphorylation cascades, this implies that Twist expression must be under a tight control at the post-translational level in order to respond in a timely manner to external stimuli.Methodology/Principal FindingsOur data show that IL-6 increases Twist expression via a transcription-independent mechanism in many SCCHN cell lines. Further investigation revealed that IL-6 stabilizes Twist in SCCHN cell lines through casein kinase 2 (CK2) phosphorylation of Twist residues S18 and S20, and that this phosphorylation inhibits degradation of Twist. Twist phosphorylation not only increases its stability but also enhances cell motility. Thus, post-translational modulation of Twist contributes to its tumor-promoting properties.Conclusions/SignificanceOur study shows Twist expression can be regulated at the post-translational level through phosphorylation by CK2, which increases Twist stability in response to IL-6 stimulation. Our findings not only provide novel mechanistic insights into post-translational regulation of Twist but also suggest that CK2 may be a viable therapeutic target in SCCHN.

Abstract 4752: Cortactin and coronin 1B cooperate to promote tumor cell invasion in head and neck squamous cell carcinoma

Abstract Amplification of chromosome 11q13 in head and neck squamous cell carcinoma (HNSCC) is a common occurrence associated with poor patient prognosis. Within the core amplified region is the gene that encodes cortactin (CTTN), a filamentous (F)- actin binding protein and Src kinase substrate that stabilizes Arp2/3-F-actin branchpoints in dynamic cortical F-actin structures. CTTN amplification results in increased lymph node metastases and decreased survival rates in HNSCC patients. CTTN amplification in HNSCC cell lines enhances tumor cell motility and invasion. Chromosomal regions of varying length flanking the core 11q13 amplicon are often amplified in HNSCC, and contain several genes with the potential to cooperate with CTTN in regulating actin dynamics. One of these genes within the 11q13 region outside of the core amplicon is CORO1B (coronin 1B), an actin-binding protein that antagonizes cortactin function by promoting the breakdown of cortactin-stabilized Arp2/3-F-actin branches, resulting in impaired cell motility. Here we show that coronin 1B is a potential mediator of HNSCC invasion. CORO1B amplification and overexpression occurs in a subset of HNSCC patients with CTTN amplification. Coronin 1B localizes with cortactin within HNSCC invadopodia, ventral membranous protrusions responsible for degrading extracellular matrix to facilitate loco-regional invasion and extracapsular spread. RNAi-mediated coronin 1B knockdown decreases the number of invadopodia per cell, enhances cell spreading and reduces extracellular matrix degradation. These results collectively point to a potential role for coronin 1B in regulating HNSCC tumor cell invasion through modulating cortactin activity and invadopodia function. Our data also support a potential diagnostic function for CORO1B amplification in denoting a previously unidentified HNSCC subset that may display increased invasiveness in cases containing 11q13 amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4752. doi:10.1158/1538-7445.AM2011-4752

Abstract 3968: Mutation and phosphorylation of p53 may switch TGF-beta from a tumor suppressor to a tumor promoter

Abstract Transforming growth factor-β (TGF-β) is a tumor suppressor during early tumor outgrowth, but becomes a tumor promoter at later stages by enhancing tumor cell motility, invasion and metastasis. Therefore, anti-TGF-β cancer therapy should exercise great caution due to the dual nature of the TGF-β signaling. However, it is still unclear when the TGF-β inhibitor should be applied to abrogate the tumor-promoting activity of TGF-β signaling. Thus, studies aiming to unravel key mechanisms by which TGF-β signaling is switched from tumor suppressor to tumor promoter will greatly benefit the design of efficient clinical treatment of cancer with TGF-β inhibitors. Recently, it was reported that the oncogenic activity of TGF-β in the MDA-MB-231 breast cancer cells was due to the presence of the mutant p53. TGF-β-induced cell migration and metastasis were enhanced by the sequestration of the tumor suppressor p63 into a ternary complex containing the mutant p53 and the intracellular TGF-β signaling mediators Smad2/3. The phosphorylation of mutant p53 at Ser6/Ser9 by Ras/MAPK signaling was essential for the formation of the ternary complex and appeared to be a crucial hallmark for the switch of TGF-β to be a tumor promoter. To better understand the molecular mechanism driving the switch of TGF-β pathway to become a tumor promoter, we performed a study using spontaneously immortalized and non-tumorigenic human mammary epithelial MCF-10A cell line (10A). We found that ectopic expression of both oncogenic H-RasV12 and mutant p53 (R175H) in 10A enhanced phosphorylation of p53 (P-p53) at Ser6/9 and TGF-β-induced formation of p53/P-Smad2 complex. Additionally, the cellular migration of those transformed 10A was more sensitive to TGF-β when compared with 10A expressing H-RasV12 alone. These results indicate that mutant p53 may induce TGF-β to become a tumor promoter in our model derived from 10A. Notably, we found there might be a PI3K/AKT signaling-dependent fashion for the switch of TGF-β to become a tumor promoter as 10A with ectopic expression of Simian virus 40 T antigens also expressed a high level of P-p53 at Ser6/9 with active PI3K/AKT signaling in contrast to active MAPK signaling. Furthermore, the level of P-p53 at Ser9 was elevated in the 10A and the p53-null PC-3 prostate cancer cells with ectopic expression of both constitutively active AKT (*AKT) and mutant p53 R175H. Importantly, TGF-β-induced interaction among p63, p53 R175H and P-Smad2 was significantly increased in the absence of a strong MAPK signaling activity in those transformed cells. Taken together, our studies may provide a novel mechanism for switching TGF-β to play the oncogenic role. In addition, we can use the model systems with key molecular alterations such as mutant p53 together with oncogenic Ras or *AKT to test the efficacy of TGF-β inhibitors as a novel cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3968.

Abstract 3826: Neutralization of TGFβ enhances the efficacy of chemotherapeutics in a preclinical model of triple negative breast cancer

Abstract TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Previously we have demonstrated that neutralizing TGFβ with the antibody 1D11 significantly inhibited metastasis in preclinical models of breast cancer and melanoma, with minimal effects on primary tumor growth. Neutralizing the protumorigenic effects that TGFβ has on the immune system and tumor microenvironment could, hypothetically, enhance the direct anti-tumor effects of chemotherapeutic agents. To test this hypothesis, several studies were conducted testing combinatorial therapies with 1D11 and chemotherapeutics against subcutaneous (SQ) 4T1, a murine model of triple negative breast cancer (TNBC), with subsequent metastasis to the lung. Paclitaxel is currently the front-line therapy for TNBC in the clinic. Combining 1D11 with paclitaxel had little effect on enhancing the efficacy of paclitaxel alone against primary tumor growth. However, the combinatorial therapy of 1D11 and paclitaxel inhibited spontaneous metastasis from the primary SQ tumor to the lung to a greater extent than either agent alone. In the clinic TNBC typically becomes resistant to taxane therapy and another treatment that has shown some promise in taxane-refractory TNBC is the combinatorial therapy of capecitabine and ixabepilone. In the 4T1 preclinical model, both capecitabine and ixabepilone inhibited the growth of SQ 4T1 tumors as single agents, and the combination of the two therapeutics resulted in slightly greater efficacy. However, the addition of 1D11 to capecitabine and ixabepilone resulted in even greater efficacy than with the combination alone. Currently, the effects of neutralizing TGFβ with 1D11 during the course of capecitabine/ixabepilone therapy on metastasis in the 4T1 model are being investigated. Additionally, mechanism of action studies are being conducted to determine the mechanisms by which 1D11 is enhancing the efficacy of paclitaxel and the combinatorial therapy. The data from these chemo-combo studies with 1D11 suggests that therapies that target TGFβ may enhance the efficacy of paclitaxel against TNBC metastasis and may potentiate the efficacy of the combinatorial therapy of capecitabine/ixabepilone in taxane-refractory TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3826.

Abstract 3431: Role of tumor microenvironment in promoting tumor metastasis

Abstract Metastatic spread of tumor cells to vital organs is the major cause of mortality in cancer patients. Tumor metastasis is a complex process involving multiple steps including detachment of tumor cells from the primary tumor, interaction with vascular endothelium and invasion of target organs. Although our understanding of the molecular and biological events that contribute to tumor cell progression has increased considerably over the last decade, we still know very little about the role of tumor-accessory cells in promoting tumor cell release from the primary tumor and metastasis to distal sites. It is well established that tumor angiogenesis plays a critical role in tumor progression. We have recently demonstrated that VEGF, in addition to its pro-angiogenic function, also induces the expression of Bcl-2 in the microvascular endothelial cells. Furthermore, tumor samples from head and neck cancer patients showed significantly higher Bcl-2 expression in tumor blood vessels and this enhanced Bcl-2 expression in tumor-associated endothelial cells was directly correlated with metastatic status of these patients. Upregulated Bcl-2 expression in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a SCID mouse model. While much is known about the role of Bcl-2 in cell survival, very little is known about the role of Bcl-2 in tumor metastasis. In this study, we examined the role and mechanism(s) by which Bcl-2 expression in tumor-associated endothelial cells may promote tumor metastasis. Our results suggest that endothelial cells expressing Bcl-2 (EC-Bcl-2) promote tumor metastasis via cell-cell interaction with tumor cells as well as by secreting chemokines including IL-6 and IL-8. EC-Bcl-2 secreted IL-6 induced EMT-related changes (downregulation of E-cadherin and upregulation of vimentin proteins) in the normal oral keratinocytes as well as head and neck tumor cells. IL-8 promoted tumor metastasis by enhancing tumor cell motility and invasiveness. In addition, EC-Bcl-2 cells exhibited significantly higher adhesion molecules expression and showed enhanced tumor cell binding. Tumor cells, when cultured alone in non-adherent conditions, showed marked increase in apoptosis (anoikis). In contrast, tumor cells showed a significant decrease in anoikis when co-cultured with EC-Bcl-2 in non-adherent conditions. Similarly, tumor cells when injected along with EC-Bcl-2 showed significantly higher lung metastasis as compared to tumor cell and EC-VC or tumor cell alone group. Taken together, these results suggest that endothelial cells expressing Bcl-2 promote tumor cell metastasis by enhancing tumor cell release and chaperoning them to distal sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3431.

Abstract 2832: Genetic variation in TGFB1, TGFBR1, TGFBR2, and prostate cancer risk and recurrence

Abstract Background: The transforming growth factor-β (TGF-β) signaling pathway has been reported to play a dual role in prostate carcinogenesis. Initially, TGF-β signaling inhibits tumor growth by its antiproliferative activity, but eventually it promotes tumor development by enhancing tumor cell motility and metastasis. We investigated whether common genetic variation in TGFB1, TGFBR1, and TGFBR2 influences the risk of aggressive prostate cancer and biochemical recurrence. Methods: Forty-four tag SNPs in TGFB1, TGFBR1, and TGFBR2 were examined in a case-control study of aggressive prostate cancer (501 cases/538 controls) and a follow-up study of biochemical recurrence (824 cases). Aggressive disease was defined as having either having a Gleason score ≥7 or TNM stage ≥ T2c, or PSA at diagnosis >10 ng/ml. Biochemical recurrence was defined as post-treatment prostate specific antigen (PSA) levels >0.3 ng/ml for radical prostatectomy patients or 2 ng/ml increase above the nadir for radiotherpay patients. Odds ratios (OR) and 95% Confidence Intervals (CI) were estimated by unconditional logistic regression to evaluate the association between SNPs and aggressive prostate cancer risk. Hazard ratios (HR) and 95% CI were estimated by Cox proportional hazard regression to examine the association between SNPs and biochemical recurrence. Results: For risk of aggressive disease, three SNPs were nominally statistically significant (TGFB1-rs4803455; TGFBR1-rs10512263; TGFBR2-rs2276768) with the most compelling polymorphism (TGFBR1-rs10512263) having an OR=1.76; 95% CI: 1.14-2.43 (P = 0.008) per additional minor allele. For risk of biochemical recurrence, seven SNPs were nominally statistical significant (TGFBR1- rs10739778; TGFBR2- rs12493607, rs2082224, rs1346907, rs1431131, rs1835538, rs11129421) with TGFBR2- rs12493607 associated with a HR=1.40; 95% CI: 1.14-1.71; P=0.001 per additional minor allele. Conclusion: Our study suggests that genetic variation in the TGF-β pathway may be associated with risk of aggressive prostate cancer and biochemical recurrence. Future work will evaluate the cumulative and interactive effects of the associations within this pathway on prostate cancer risk and recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2832.

Relaxin Enhances S100A4 and Promotes Growth of Human Thyroid Carcinoma Cell Xenografts

Relaxin increases cell motility and in vitro invasiveness in human thyroid carcinoma cells but the underlying molecular mechanisms of this action are largely unknown. In the present study, we show that relaxin transcriptionally upregulates the calcium-binding protein S100A4 (metastasin) and increases the cytosolic 10-kDa monomer and the 20-kDa dimer form of S100A4 in human thyroid carcinoma cells. The relaxin-induced increase in cell motility was blocked completely when S100A4 expression was diminished using an S100A4 small interfering RNA knockdown approach. We have shown previously the expression of the insulin-like family member relaxin in human thyroid carcinoma tissues but not in benign thyroid tissues. Human thyroid carcinoma tissues expressing relaxin also stained positive for S100A4. In nude mouse experiments, human thyroid carcinoma cell transfectants with constitutive expression of relaxin generated large and fast-growing tumors with significantly increased numbers of proliferating cells. We provide evidence in our cell model that the relaxin target protein S100A4 secreted by the thyroid carcinoma transfectants may not only enhance tumor cell motility but also promote xenograft angiogenesis as determined by the higher density of tumor microvessels and the angiogenic potential of S100A4 in in vitro tube formation assays. In conclusion, we have identified S100A4 as a major mediator of the actions of relaxin in thyroid carcinoma cell motility and in vivo thyroid tumor angiogenesis.

Abstract B244: Inhibition of metastases by a neutralizing TGFβ-specific antibody involves the activity of cytotoxic T lymphocytes and natural killer cells

Abstract TGFβ plays a key role in metastasis by promoting epithelial to mesenchymal transition, up-regulating proteases that degrade extracellular matrix, enhancing tumor cell motility and suppressing antitumor immunity. Preclinical studies were conducted to determine the effect of neutralizing TGFβ on both primary tumor growth and metastases in in vivo models of experimental and spontaneous metastasis. Treatment with the pan-neutralizing TGFβ antibody, 1D11, as a single agent had limited effect on the growth of established, primary, syngeneic tumors (4T1 mammary carcinomas or B16-F10 melanomas) and did not enhance the efficacy of chemotherapeutic agents against these primary tumors. However, TGFβ antagonism was shown to have a significant effect on inhibiting metastasis to the bone following intracardiac injection of 4T1 cells. In addition, it was demonstrated that neutralization of TGFβ in B16-F10 melanoma models inhibited experimental pulmonary metastases following injection of cells into the tail vein, and inhibited spontaneous metastases from a subcutaneous primary tumor to the draining lymph node. Studies aimed at elucidating the mechanism of action of 1D11 in mice lacking specific immune effector cells suggest that the activity of cytotoxic T lymphocytes is important in the inhibition of metastases. The role of natural killer (NK) cells is less clear; NK cell activity is not required for inhibition of experimental metastasis to the lung, but is critical for the ability of 1D11 to inhibit spontaneous metastasis from a primary tumor to the draining lymph node. Investigation into the effects of neutralizing TGFβ on innate immunity and the tumor microenvironment are currently ongoing. Together these data suggest that neutralization of TGFβ significantly inhibits metastasis and anti-TGFβ strategies may be effective at treating metastatic disease in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B244.

High Expression of Osteopontin and CD44v6 in Odontogenic Keratocysts

Odontogenic keratocysts (OKCs) are more aggressive and more osteolytic lesions than dentigerous cysts (DCs) and radicular cysts (RCs). Osteopontin (OPN) is related to cancer metastasis and bone destruction. Binding of OPN to its cell membrane receptors integrin alphav and CD44v6 can enhance tumor cell motility, migration, invasion and spread. This study assessed the possible contribution of OPN, integrin alphav and CD44v6 to the local aggressive behavior and osteolytic ability of OKCs. We used an immunohistochemical method to examine the expression of OPN, integrin alphav and CD44v6 in tissue sections of 20 OKCs, eight DCs and 10 RCs. We found strong cytoplasmic OPN immunostaining in lining epithelial cells of 8 of 20 OKCs but not in any DCs and RCs. Positive OPN staining was also noted in the subepithelial connective tissue of four OKCs with intraepithelial expression of OPN. Diffuse and strong membranous integrin alphav staining was discovered in osteoclasts in all our tissue sections and in nearly all lining epithelial cells of DCs and RCs, but not in OKCs. In addition, diffuse and strong membranous CD44v6 staining was also observed in nearly all lining epithelial cells of OKCs, DCs and RCs. Binding of OPN to osteoclast cell membrane receptor integrin alphav can activate the osteoclasts and increase their osteolytic activity. In addition, binding of OPN to OKC lining epithelial cell membrane receptor CD44v6 can enhance the motility, migration, invasion and spread of lining epithelial cells into the surrounding cancellous bone. Therefore, we suggest that the local aggressive behavior and high osteolytic ability of OKCs in the jawbone can be explained at least partially by high expression of OPN and CD44v6 in lining epithelial cells of OKCs and high expression of integrin alphav in osteoclasts.

Fascin overexpression correlates with positive thrombospondin-1 and syndecan-1 expressions and a more aggressive clinical course in patients with gallbladder cancer

This study was conducted to evaluate the prognostic role of fascin expression in gallbladder (GB) cancer and to define the relationship of thrombospondin-1 (TSP-1) and syndecan-1 in fascin expression. We performed immunohistochemical detection of fascin, TSP-1, and syndecan-1 in 43 tissue samples from GB cancer patients who underwent macroscopic complete resection. There were 19 (44%) and 24 (56%) cases having low- and high-grade fascin expression, respectively. The tumors with high-grade fascin expression tended to more frequently show poorer differentiation, deeper invasion depth, lymph node metastasis, a higher American Joint Committee on Cancer stage, and recurrence (each P < 0.05). The patients with high-grade fascin expression had significantly shorter survival periods than those with low-grade fascin expression (P < 0.05). The frequency of positive TSP-1 or syndecan-1 expression in the cases with high-grade fascin expression was significantly higher than that in the cases with low-grade fascin expression (each P < 0.05). These results suggest that a subset of advanced GB cancers revealed a marked overexpression of fascin, which was associated with aggressive clinicopathologic findings and poor overall survival. Furthermore, fascin, TSP-1, and syndecan-1 may act in concert to mediate a more aggressive clinical course through enhanced tumor cell motility.

The cellular localization of autotaxin impacts on its biological functions in human thyroid carcinoma cells

Autotaxin (ATX/NPP2) shows a nucleotide pyrophosphatase/phosphodiesterase and lysophospholipase D (lysoPLD) activity and is a member of a family of structurally-related mammalian ecto-nucleotide pyrophosphate/phosphodiesterases (E-NPP1-3). ATX is unique among E-NPP as it is secreted and not membrane-bound as are NPP1 and -3. The ATX gene activity is significantly higher in undifferentiated anaplastic (UTC) as compared to follicular (FTC) and papillary thyroid carcinomas (PTC) or goiter tissues. ATX also enhances the motility of thyroid tumor cells. We bio-engineered stable transfectants of the human thyroid carcinoma cell line FTC-238 expressing either bioactively-secreted (sATX) or membrane-anchored ATX (mATX) to identify the biological functions of ATX which critically depend on the E-NPP member being secreted and provide insight into the effects of high local ATX concentrations and cellular responses. An increased cell motility was exclusively observed with FTC-238 sATX transfectants, whereas membrane-anchored ATX appeared to impair motility. We identified IL-1beta as an upstream suppressor of ATX expression in FTC-238, ATX-mediated motility in FTC-238 and stable transfectants, with IL-1beta having the strongest motility-suppressive effect on FTC-238 sATX clones. sATX and mATX strongly increased the anchorage-independent colony formation of FTC-238 but the size and number of colonies formed in the soft agar were significantly smaller in FTC-238 mATX versus the FTC-238 sATX clones. The cancer-testis antigen BAGE was identified as a novel target gene of ATX in FTC-238. Transcript levels for BAGE were 6-fold higher in FTC-238 mATX versus sATX clones. Increased BAGE transcript levels were also detected in tissues of patients with UTC versus FTC, PTC or goiter tissues. In summary, enhanced tumor cell motility and tumorigenic capacity critically depended on sATX in thyroid carcinoma cells. Irrespective of its compartmentalization, the cancer-testis antigen BAGE was identified as a novel target gene of ATX in FTC-238 and a potential new tissue marker in UTC tissues, which we had previously shown to express high levels of ATX.

INSL3 in the benign hyperplastic and neoplastic human prostate gland

The human prostate gland is a well known source of H1 and H2 relaxin but information is lacking on the expression and potential role of the INSL3 peptide hormone within the prostate gland. In the present study we have investigated the expression of human INSL3 in patients with benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma tissues. Of the prostate epithelial cells, strongest INSL3 expression was detected in the basal epithelial cell compartment. Weaker INSL3 mRNA expression and immunoreactive INSL3 production were observed in secretory epithelial cells and in interstitial smooth muscle cells. Prostate epithelial cells were also a source for transcripts encoding the INSL3 receptor LGR8 suggesting the presence of an autocrine/paracrine INSL3-LGR8 ligand-receptor system within the human prostate. Three human prostate carcinoma cell lines displayed differential gene activity for INSL3 and LGR8. While LNCaP was devoid of INSL3, the androgen-insensitive PC-3 and the stromal prostate cell line hPCP co-expressed INSL3 and LGR8 transcripts. In addition to expressing INSL3 mRNA, the LGR8-negative DU-145 also expressed an INSL3 splice form formerly demonstrated in thyroid carcinoma cells. When incubated with recombinant INSL3, PC-3 cells showed significantly increased intracellular cAMP levels indicating functional LGR8 receptors. INSL3 did not alter the proliferative or metabolic activity of PC-3 carcinoma cells. Instead, PC-3 responded to INSL3 with significantly enhanced tumor cell motility and a transcriptional down-regulation of ErbB receptors and EGF. All-trans-retinoic acid was demonstrated in PC-3 to up-regulate LGR8 gene activity in a dose- and time-dependent manner while having no effect on INSL3 gene activity. In conclusion, we have identified a functional INSL3-LGR8 ligand-receptor system in human prostate carcinoma cells.

Galectin-1 knocking down in human U87 glioblastoma cells alters their gene expression pattern

We have previously reported that (i) progression of malignancy in patients bearing astrocytic tumors correlates with increased tumor levels of galectin-1; (ii) in vitro addition of purified galectin-1 to U87 human glioblastoma cells enhances tumor cell motility; and (iii) conversely, knocking down galectin-1 expression in this cell line by stable transfection with antisense galectin-1 mRNA impairs motility and delays mortality after their intracranial grafting to nude mice. We here used cDNA microarray analysis to compare the effect on gene expression of stable transfection with antisense galectin-1 vector to mock-transfected and wild-type cells. Among the 631 spots probing genes potentially involved in cancer that were valid for analysis on all the arrays the expression of 86 genes was increased at least 2-fold. Confirmation of increased protein levels was provided by immunocytochemistry for p21 waf/cip1, cullin-2, p53, ADAM-15, and MAP-2. Major differences in the expression patterns of ADAM-15 and the actin stress fiber organization were also observed. U87 cells stably deficient for galectin-1 expression were significantly less motile than control. We conclude that the stable inhibition of galectin-1 expression alters the expression of a number of genes that either directly or indirectly influence adhesion, motility and invasion of human glioblastoma cells.

Epidermal growth factor receptor dependence in human tumors: more than just expression?

The epidermal growth factor receptor (EGFR) is a rational target for antitumor strategies. EGFR signaling causes increased proliferation, decreased apoptosis, and enhanced tumor cell motility and neo-angiogenesis. The EGFR is expressed or highly expressed in a variety of human tumors of epithelial origin. ZD1839 (Iressa) is an orally active, selective EGFR tyrosine kinase inhibitor, which blocks signal transduction pathways implicated in proliferation and survival of cancer cells. The lack of a consistent method of evaluating levels of EGFR has caused a disparity in reports of the EGFR as a prognostic factor; however, for some tumors, EGFR is a strong prognostic indicator associated with more aggressive disease and reduced survival. So far, no clear association between EGFR levels and response to EGFR-targeted agents has been found. Preclinical studies with ZD1839 have noted a relationship between the two in some cases, but not others. EGFR signaling may be increased by a number of mechanisms in addition to high expression levels of EGFR, including receptor mutations, heterodimerization with other members of this receptor family such as HER2 (erbB2), increased expression of (autocrine/ paracrine) ligands, and alterations in molecules that control receptor signaling output. Each of these components could be assessed to give an indication of the magnitude of EGFR signal amplification. Evaluation of signaling components downstream from EGFR should provide information on the activation of the EGFR pathway. Until EGFR-based assays predictive of a response to receptor-targeted therapies are available, there is no clear justification for stratifying patients by EGFR status or excluding patients with low EGFR levels from trials with ZD1839 or other EGFR inhibitors.

Keratinocyte growth factor-induced motility of breast cancer cells.

Endogenous growth factors and cytokines are known to have a major influence on the progression, motility and invasiveness of tumor cells. We have reported previously that conditioned media from mouse fibroblasts increases the motility of breast cancer cells. Further, we determined that keratinocyte growth factor (KGF) was an active factor from mouse fibroblasts responsible for most of the motility response in breast cancer cells. The present study examined the effect of Human KGF on the motility of estrogen receptor (ER)-positive and ER-negative human breast cancer cell lines in culture using time-lapse videomicroscopy to quantify cell motility. In the present study we observed that recombinant human KGF enhanced several parameters of cellular motility in ER-positive cells but not in ER-negative cell lines. Further, we observed that the level of KGF receptor (KGFR) expression in ER-positive cells was much greater than in the ER-negative cell lines. The motility response to KGF was found to be both dose-and time-dependent. Of the three ER-positive breast cancer cell lines tested. MCF-7 cells were the most responsive to KGF stimulation. Finally, MCF-7 cells grown in estrogen-depleted media did not respond to KGF. These results suggest that KGF from stromal tissue surrounding a primary tumor mass can enhance tumor cell motility and may be an early signal in the progression of breast cancer cells to a more motile and metastatic phenotype. Thus, KGF, KGFR and/or the KGF signaling pathway may be important therapeutic targets for the treatment or prevention of breast cancer metastasis.

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion.

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.

Chemotactic response of rat mammary adenocarcinoma cell clones to tumor-derived cytokines

A cytokine with an apparent molecular weight of 53,000 daltons was isolated from serum-free medium conditioned by MTLn3 cells or from homogenates of MTLn3 cells, a highly metastatic variant of the rat 13762NF mammary adenocarcinoma. The chemotactic responses of MTLn3 and the low metastatic variant MTLn2 cells to this cytokine were tested in vitro using modified Boyden chambers. Both the chemotactic and chemokinetic movements of MTLn3 cells were stimulated by the MTLn3-derived cytokine. In addition, the MTLn3-derived cytokine stimulated a relatively small, but significant chemotactic migration of MTLn2 tumor cells, while these cells did not respond to medium conditioned by MTLn2 cells. MTLn3 cells themselves did not respond chemotactically to type I collagen or medium conditioned by MTLn2 cells. These results suggest that the chemotactic response may be a function of metastatic potential of the invading tumor cells. The production of tumor cytokines that enhance tumor cell motility may thus represent a phenotypic difference between 13762NF tumor cell subpopulations of high and low metastatic potential.