Anti-metastatic action of an N 4-aryl substituted thiosemicarbazone on advanced triple negative breast cancer.

PURPOSE: Advanced triple negative breast cancer (ATNBC) is defined by a lack of expression of hormones receptors as well as HER2/neu and its high probability of visceral metastasis. This pathology is associated with a poor prognosis. Previously, we found that T2, an N 4-arylsubstituted thiosemicarbazone (N 4-TSC), had cytotoxic effect on human breast cancer cells lines. Hence, in this study, we investigated the anti-metastasic action of T2 on ATNBC. METHODS: In order to deepen T2 action mode on ATNBC, we first confirmed T2 cytotoxicity on a panel of TNBC cells and then continued studying T2 effects in vitro an in vivo on the syngeneic 4T1 mouse model. RESULTS: We found that T2 had a cytotoxic effect comparable to chemotherapeutics used in present treatment schemes for ATNBC. T2 treatment not only induced apoptosis, but it also down-modulated 4T1 invasive and metastatic-associated capacities, such as clonogenicity, migration and metallo-proteases activity. Moreover, this agent reduced the number of 4T1 cancer stem cells. Finally, T2 treatment induced a more differentiated cell phenotype and the overexpression of the metastasis suppressor gene NDRG-1. In vivo assays showed that T2 reduced tumor burden, down modulated local tumor invasion and significantly reduced the number of lung metastases in the 4T1 advanced TNBC murine model, while the compound did not exhibit intolerable toxicity. CONCLUSION: This study provided evidence that T2 not only exerted an anti-tumor activity but it also showed anti-invasive and anti-metastatic actions on ATNBC in vivo and in vitro, suggesting that T2 could be considered as a promising therapy that deserves further analysis.

Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.

Glypican-3 reexpression regulates apoptosis in murine adenocarcinoma mammary cells modulating PI3K/Akt and p38MAPK signaling pathways.

Glypican-3 (GPC3) is a proteoglycan involved in proliferation and cell survival. Several reports demonstrated that GPC3 is downregulated in some tumors, such as breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their invasive and metastatic capacities, associated with a decrease of their motility and an increase of their cell death. We demonstrated that GPC3 inhibits canonical Wnt signaling, as well as it activates non canonical pathway. Now, we identified signaling pathways responsible for the pro-apoptotic role of GPC3 in LM3 cells. We found for the first time that GPC3 inhibits the PI3K/Akt anti-apoptotic pathway while it stimulates the p38MAPK stress-activated one. We report a concomitant modulation of CDK inhibitors as well as of pro- and anti-apoptotic molecules. Our results provide new clues regarding the mechanism involved in the modulation induced by GPC3 of mammary tumor cell growth and survival.

Inhibition of invasion and metastasis by glypican-3 in a syngeneic breast cancer model.

Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and beta-catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF-beta increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.

GM-CSF secreted by murine adenocarcinoma cells modulates tumor progression and immune activity.

During tumor development, growth factors may act in autocrine manner stimulating cell proliferation, or in paracrine manner affecting the microenvironment of the tumor and modulating the immune system. Murine mammary adenocarcinoma M3 tumor bearers develop lung metastases and leukocytosis during its evolution. Previously we described that M3 conditioned media enhanced metastasis incidence, when it was inoculated in tumor-operated mice. In the present study we determine that spleen cells from M3 tumor operated mice treated with M3 conditioned media, were able to transfer the capacity to enhance metastasis to other tumor operated mice. Spleen cells have immune suppressor activity that could be reversed by cyclophosfamide treatment. M3 tumor cells secrete GM-CSF, which is able to promote in vitro proliferation of M3 cells as well as spleen cells. This proliferation could be abrogated by the addition of anti-GM-CSF. We report that the GM-CSF secreted by M3 tumor cells had stimulatory activity on M3 tumor cell and lymphocyte proliferation.

RalA mediates v-Src, v-Ras, and v-Raf regulation of CD44 and fibronectin expression in NIH3T3 fibroblasts.

Oncogenic transformation of fibroblasts by v-Src and v-Ras is often associated with downregulation of fibronectin (FN) and increased expression of CD44, a receptor for hyaluronan. Both v-Src and v-Ras as well as v-Raf activate phospholipase D through the small GTPase, RalA, an important mediator of transformation and tumorigenesis in vivo. We have therefore investigated whether RalA is involved in the downregulation of FN and overproduction of CD44 upon oncogenic transformation. We report here that compared to untransfected cells NIH3T3 cells transformed by v-Src, v-Ras, or v-Raf have reduced levels of FN and increased levels of CD44. Moreover, the ability to form extracellular FN fibrils was significantly reduced in the oncogene-transformed cells compared to parental controls. Coexpression of the dominant negative S28N-RalA mutant restored the levels of CD44 and FN and the capacity of v-Src-, v-Ras-, and v-Raf-expressing cells to form extracellular FN fibrils, to those observed in NIH3T3 cells. The data presented here show a novel regulatory role for RalA, which is required for tumor formation in transformed NIH3T3 cells, in mediating the signal transduction pathway activated by v-Src, v-Ras, and v-Raf, that leads to FN downregulation and CD44 overexpression.

Apoptotic cell death in mammary adenocarcinoma cells is prevented by soluble factors present in the target organ of metastasis.

Target organ of metastasis determines the fate of metastasis. The soluble factors released from one or more cell types in the new stroma may influence growth and survival of metastatic cells. In the present study, we used conditioned media from the kidney, liver and lung, the latter being the target organ of metastasis of murine mammary adenocarcinoma cell lines LM3, LMM3 and F3II, to assess whether the soluble factors released from these organs could modulate in vitro survival of these cell lines after apoptosis-inducing treatments and to investigate the mechanisms involved in this effect. We demonstrate that conditioned medium from lung, but not from liver or kidney, promotes survival of these cells after doxorubicin, cisplatin, agonistic anti-Fas antibody and serum withdrawal treatments. Furthermore, LMM3 cells treated with lung conditioned medium after doxorubicin exposure maintained their tumorigenic capacity and metastatic potential. Neither IGF nor EGF could promote survival but, surprisingly, TGF-beta could reduce sensitivity of LMM3 cells to doxorubicin in vitro. Doxorubicin treatment induced Bax expression and down-regulated Bcl-2 expression. In contrast, lung conditioned medium increased Bcl-2 expression and inhibited doxorubicin-mediated Bcl-2 down-regulation. Neither of those treatments alone modified Bcl-X(L) expression, although co-treatment induced a 3- to 5-fold increase of its expression. These results suggest that the lung microenvironment could promote metastasis of these adenocarcinoma cell lines by increasing survival of metastatic cells, possibly by modulation of Bcl-2 protein family expression.

Cathepsin B levels in urine from bladder cancer patients.

There is accumulating evidence that cysteine proteinase activity plays an important role in cancer cell invasion and metastasis. Previously we demonstrated that cathepsin B (CB) plasma activity is increased in patients with transitional bladder cancer (TCC). In this work we have attempted to determine whether urine CB protein levels could be used as tumor marker in bladder cancer patients. Urine CB levels were evaluated employing a dot blot method, in 30 patients with TCC, 21 patients successfully treated from TCC without evidence of disease at the moment of urine collection (NED) and in 30 healthy volunteers. The median value (Md) of the control group was 3.8 microg CB/ml. Significantly higher urine CB values (Md: 5.9 microg/ml) were found in the TCC group. A high CB value was also found in the NED group (5.0 microg/ml). Urine CB values over the 5.2 microg/ml (cut-off point) were observed in 63% of TCC patients, 48% of NED and 8% of the control group. Only 4% NED patients had CB values over 13.0 microg/ml while 33% of TCC patients surpassed this value. Thus, urine CB might be a potential marker for transitional bladder cancer diagnosis.

Plasma metalloproteinase activity is enhanced in the euglobulin fraction of breast and lung cancer patients.

Matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis. We verified, by gelatin zymography, MMP activity in the euglobulin plasma fraction of 82 healthy controls, 66 patients with benign diseases and 149 patients with breast, lung, colon or brain cancer. The euglobulin fractions assayed showed 4 gelatinolytic bands of 62, 92, 120 and 200 kDa. The median (Md) value for 92 kDa-MMP activity was significantly increased in breast (Md 1.34 arbitrary units [AU]/ml plasma, range 0.0-7.2) and lung cancer patients (Md 1.43 AU/ml, range 0.0-3.6) compared with the controls (Md 0.48 AU/ml, range 0.0-1.8). Patients with colon cancer or gliomas presented values of MMP-9 similar to those of the healthy population. Multivariate analysis indicated that plasma MMP-9 activity was not predicted by the known clinicopathological parameters such as age, stage, tumor size, number of positive lymph nodes, histologic grade, histologic type, nuclear grade or mitotic index. Lung cancer patients also presented high values of MMP-9 (Md 1.43, range 0.0-3.6 [n = 26]), without association with tumor stage or histologic type. The levels of 92 kDa-MMP activity in the plasma euglobulin fraction could be a potentially useful tumor marker in breast and lung cancer.

Soluble factors from the target organ enhance tumor cell angiogenesis: role of laminin SIKVAV sequence.

The ability of tumor cells to respond to microenvironmental factors present in the target organ determines in part the successful development of a metastasis. In a previous work it was demonstrated that the conditioned medium (CM) from lungs of normal mice stimulates in vitro migration, proliferation and uPA activity of cells from a murine mammary adenocarcinoma moderately metastatic to lung. This CM also enhanced local and metastatic tumor growth. Here, we show that lung CM enhanced neovascularization when inoculated together with LM3 tumor cells into the skin of syngeneic mice. A similar tumor-induced angiogenesis response was obtained when lung CM was injected systemically. Western blot analysis of lung CM revealed the presence of some laminin fragments containing the sequence SIKVAV. To determine whether those molecules were responsible for the observed angiogenic effects, the CM was depleted of the peptides containing the SIKVAV sequence. We observed that the SIKVAV-depleted lung CM lost its ability to induce an enhancement of the tumor neovascular response. Our results suggest a role for the target organ in facilitating the neovascularization of tumor cells, probably through the participation of active peptides derived from the proteolytic degradation of the basement membrane component laminin.

Antimetastatic effect of desmopressin in a mouse mammary tumor model.

We have investigated the effects of desmopressin (DDAVP), a synthetic analog of the natural hormone vasopressin, on experimental lung colonization of mammary tumor cells using a syngeneic BALB/c mouse model. Coinjection of DDAVP (1-2 microg/kg body weight) at the time of i.v. inoculation of F3II carcinoma cells or LM3 adenocarcinoma cells significantly inhibited the formation of experimental lung metastases. In both cases, the number of pulmonary nodules was reduced about 70%. Inhibition of metastasis was also obtained with i.v. administration of DDAVP 24 h after tumor cell inoculation. Interestingly, the inhibition of lung metastasis was not due to direct cytotoxic effects of DDAVP on mammary tumor cells. The in vitro formation of multicellular aggregates in the presence of citrated plasma from control and DDAVP-treated mice was also examined. Control plasma rapidly induced a significant tumor cell aggregation. In contrast, in the presence of plasma from DDAVP-treated mice, tumor cells remained as a single cell suspension. DDAVP may help to dissolve the protective fibrin shield of circulating tumor cells. Our data suggest, for the first time, that adjuvant DDAVP therapy may impair successful implantation of circulating mammary tumor cells.

Verapamil inhibits tumor protease production, local invasion and metastasis development in murine carcinoma cells.

The invasion and metastasis process involves degradation of the extracellular matrix mediated by tumor- and host-produced proteolytic enzymes. The main enzymes involved in this process are urokinase-type plasminogen activator (uPA) and the matrix metalloproteinases (MMPs). Calcium is a main co-factor in the signaling pathways that regulate cell proliferation and protease production. We have studied here the effect of verapamil, a calcium channel blocker widely used to treat hypertensive diseases, on local tumor growth, spontaneous and experimental metastasis development, tumor-associated protease production and circulating MMP activity in tumor-bearing mice. BALB/c mice treated for 45 days with verapamil showed no toxic effects. Oral administration of verapamil to mice injected with F311 tumor cells, either pre-treated or not with verapamil, showed a significant decrease of local tumor invasion and both spontaneous and experimental metastasis development (51.3% inhibition of metastasis in both cases, p < 0.01). uPA and MMP-9 production by tumor cells in vitro was significantly inhibited by verapamil in a dose-dependent manner, showing a long-term inhibition after removal of the drug. Verapamil also exhibited a marked cytostatic effect on F311 cell proliferation in vitro. In addition, circulating MMP activity, usually enhanced in tumor-bearing mice, diminished significantly with all verapamil treatments. Our results suggest that modulation of the calcium-dependent signaling pathways that regulate tumor- or host-dependent production of proteases and tumor cell proliferation could contribute to the inhibition of metastasis development. Finally, we describe the inhibitory effects of a commonly used hypotensor in humans, verapamil, on the invasive and metastatic capacity of mammary tumor cells.

Expression of RGD minus fibronectin that does not form extracellular matrix fibrils is sufficient to decrease tumor metastasis.

Fibronectin (FN) is a plasma and extracellular matrix (ECM) glycoprotein, the expression of which may modulate the invasive and metastatic abilities of cancer cells. LMM3 is a cell line derived from the highly metastatic mouse mammary adenocarcinoma MM3 and is unable to express FN both at protein and mRNA levels. To study the role of FN in the metastatic process, LMM3 cells were stably transfected with 2 variants (wt and RGD-minus) of a full length human FN cDNA. All analyzed clones secreted recombinant FN and although none assembled FN in the ECM they showed an in vitro reduced migratory ability and an increased adhesive capacity. FN-producing cells were assayed for experimental and spontaneous metastasis. All clones tested showed a significant reduction in the number of experimental lung metastasis when compared with a control clone. Similar trends were observed for spontaneous metastatic ability. Our results indicate that the expression of FN that lacks the well-recognized RGD cell-binding site and that does not form ECM fibrils, is sufficient to decrease the metastatic potential of cancer cells. Our results also suggest that an RGD-independent mechanism may be acting in the prevention of metastasis.

Function and expression of CD44 during spreading, migration, and invasion of murine carcinoma cells.

The cell surface glycoprotein CD44 is proposed as a main participant in cell adhesion and migration. We studied the function, expression, and distribution of CD44 in the invasive and metastatic F3II murine carcinoma cell line during adhesion, spreading, migration, and invasion. A mAb anti-CD44 (KM 201) dramatically blocked F3II cell adhesion on both plastic and hyaluronic acid coatings, as well as spreading on uncoated plastic surfaces (P < 0.01). KM201 mAb significantly inhibited F3II cell migration and invasion in Transwell chambers. Immunocytochemistry of spreading cells revealed that CD44 distributed in bands on the cell surface, particularly in the tip of leading edges and in the perinuclear zones of the cell membrane. CD44 antigen was never detected in filopodia or lamellipodia nor in focal adhesion-like structures, but was also detectable as strong interlamellar bands. Fully spread cells showed a decreased CD44 signal compared to cells in early stages of spreading. This decrease correlated with a reduced expression of CD44 as detected by Western blot. We also investigated the signals that may regulate CD44 expression in F3II cells. Treatment of F3II cells, with phorbol myristate acetate (PMA) or phosphatidic acid (PA, the product of PLD-dependent hydrolysis of phosphatidylcholine), significantly enhanced CD44 expression. Conversely, the treatment of F3II cells with H7, a specific PKC inhibitor, or propranolol, which blocks PA conversion to DAG, significantly decreased CD44 expression levels. These results suggest the involvement of PKC and PLD pathways in CD44 expression. These results demonstrate that CD44 plays an important role during F3II cells adhesion, spreading, migration, and invasion. In addition we provide information linking the PLD- and PKC-dependent pathways with the regulation of CD44 expression.

Secretion of urokinase and metalloproteinase-9 induced by staurosporine is dependent on a tyrosine kinase pathway in mammary tumor cells.

Urokinase-type plasminogen activator (uPA) is a key serine protease involved in invasion and metastasis. We had shown that overproduction of uPA in tumor cells is controlled by a phospholipase D-protein kinase C-dependent pathway. Now we studied whether other signaling pathways participate in the regulation of constitutive uPA and metalloproteinase (MMP) overproduction in tumor cells. Staurosporine, a protein kinase inhibitor, stimulated uPA and MMP-9 secretion as measured by radial caseinolysis, zymography and Western blotting. Genistein, a specific tyrosine kinase inhibitor, reduced the constitutive and staurosporine-induced uPA and MMP-9 secretion. Interestingly, the phosphatase inhibitor vanadate stimulated uPA secretion. Verapamil, a calcium channel blocker, inhibited both endogenous and PMA-stimulated secretion of uPA but was unable to inhibit staurosporine-induced secretion. The alcohol n-butanol, a phospholipase D and protein kinase C inhibitor, besides inhibiting constitutive uPA secretion, blocked staurosporine-induced secretion. Our results suggest that constitutive and staurosporine-induced uPA and MMP-9 secretion by LM3 murine mammary tumor cells is controlled by an endogenous tyrosine kinase pathway and probably involves protein phosphatases. In addition, the staurosporine-induced signal regulating urokinase secretion is independent of extracellular calcium but dependent on phospholipase D.

Downregulation of fibronectin transcription in highly metastatic adenocarcinoma cells.

Silencing of fibronectin (FN) expression seems to be one of the key mechanisms underlying metastatic behaviour. An inverse correlation exists between FN expression levels and the metastatic potential of two related murine mammary adenocarcinomas, M3 and MM3. Primary cultures of M3 tumour, which is moderately metastatic to lung (40% incidence), show a conspicuous FN extracellular matrix (ECM) and high levels of FN mRNA, while primary cultures of the highly metastatic MM3 tumour (95% lung incidence) are negative for FN in immunofluorescence and show at least 40-fold lower levels of FN mRNA, only detectable by RT-PCR, with a different pattern of alternatively spliced EDI isoforms compared to M3 cells. We show that the FN promoter sequence is not altered in MM3 cells. Transfection experiments with CAT constructs indicate that silencing occurs at the transcriptional level, involving the 220-bp proximal promoter region.