Silencing of hyaluronan synthase 2 suppresses the malignant phenotype of invasive breast cancer cells

Accumulation of hyaluronan has been demonstrated in the peritumoral breast cancer stroma and nests of tumor cells. In this study, we have quantified the production of hyaluronan and the expression of mRNAs encoding hyaluronan synthesizing (HAS) and hyaluronan degrading (HYAL) enzymes in a panel of breast cancer cell lines. The analysis revealed that highly invasive breast cancer cells produce high amounts of hyaluronan and express preferentially HAS2 mRNA, whereas less invasive breast cancer cells produce low amount of hyaluronan and express HAS1 and HYAL1 mRNAs. We explored the importance of HAS2 expression for breast cancer tumorigenicity, by specifically silencing the HAS2 gene using RNA interference (RNAi)-mediated suppression in the invasive breast cancer cell line Hs578T. This led to a less aggressive phenotype of the breast tumor cells, as assessed by cell growth, both in anchorage-dependent and anchorage-independent cultures. siRNA-mediated knock down of HAS2 in Hs578T breast tumor cells led to an up-regulation of HAS1, HAS3 and HYAL1 mRNAs, resulting in only a 50% decrease in the net hyaluronan production; however, the synthesized hyaluronan was of lower size and more polydisparse compared to control siRNA-treated cells. Interestingly, Hs578T cells deprived of HAS2 migrated only half as efficiently as HAS2 expressing cells through cell-free areas in a culture wounding assay and through Transwell polycarbonate membrane as well as invaded a Matrigel layer. These results imply that alterations in HAS2 expression and endogenously synthesized hyaluronan affect the malignant phenotype of Hs578T breast cancer cells.     

Lipocalin 2 promotes lung metastasis of murine breast cancer cells

Background

Lipocalin 2, an iron binding protein, is abnormally expressed in some malignant human cancers and may play an important role in tumor metastasis. However, the roles of lipocalin 2 in breast cancer formation and metastasis have not been clearly shown. This study aimed to investigate the roles of lipocalin 2 in breast tumor metastasis.

Methods

Lipocalin 2 was overexpressed in the metastatic 4T1 murine mammary cancer cells. The effects of lipocalin 2 overexpression on the malignancy of breast cancer cells were examined using cell proliferation assay, migration assay, invasion assay, and soft agar assay in vitro. Tumor formation and metastasis abilities were examined using a well established mouse mammary tumor model in vivo.

Results

Lipocalin 2 overexpression significantly enhanced the migration and invasion abilities of 4T1 cells in vitro, and lung metastasis in vivo. But overexpression of lipocalin 2 in 4T1 cells didn''t affect cell proliferation and anchorage-independent growth in vitro, and primary tumor weight in vivo. Further studies demonstrated that the inhibition of the PI3K/Akt pathway could be a causative mechanism for the promotion of breast cancer migration/invasion induced by lipocalin 2 overexpression.

Conclusion

These results clarified that lipocalin 2 could promote lung metastasis of 4T1 cells through the inhibition of the PI3K/Akt pathway, suggesting that lipocalin 2 was a potential target for therapy of breast cancer.     

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis

Core 3‐derived glycans, a major type of O‐glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy because of loss of expression of functional β3‐N‐acetylglucosaminyltransferase‐6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re‐expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. Therefore, we re‐expressed core 3 synthase in human pancreatic cancer cells (Capan‐2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared to vector control cells. Expression of core 3 O‐glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the downregulation of expression of several genes including REG1α and FGFR3 and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor‐associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3‐derived O‐glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.     

Silencing of CXCR4 blocks breast cancer metastasis

RNA interference technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. For the first time in cancer research, we show that direct injection of a pool of naked small interfering RNA (siRNA) duplexes can prevent tumorigenesis in an animal model, suggesting a novel preventive and therapeutic strategy for cancer management. As a model system, we used siRNA duplexes of CXCR4 to block breast cancer metastasis. Here, we show that blocking CXCR4 expression at the mRNA level by a combination of two siRNAs impairs invasion of breast cancer cells in Matrigel invasion assay and inhibits breast cancer metastasis in an animal model. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments. Moreover, our studies confirm the necessity of CXCR4 in breast cancer metastasis.     

Silencing of connexin 43 suppresses invasion, migration and lung metastasis of rat hepatocellular carcinoma cells

To reduce cancer mortality, understanding of mechanisms of cancer metastasis is crucial. We have established six rat hepatocellular carcinoma (HCC) cell lines, which exhibit differing metastatic potential to the lung after inoculation into the tail veins of nude mice. In the present experiment, we investigated the process of cell attachment to metastatic sites and possible regulating factors. One hour after inoculation, two of two HCC cell lines with high metastatic potential and one of two HCC cell lines with low metastatic potential exhibited many attached cells in the lung. One day after inoculation, lung metastatic foci were observed only with highly-metastatic cells with elevated connexin 43 (Cx43) expression as assessed by cDNA array analysis. Furthermore, 24 or 48 h after transfection of an siRNA targeting Cx43, in vitro invasion and migration were suppressed by 68% (P < 0.001) and 36% (P < 0.05) compared with control-siRNA transfected cells, despite no differences in cellular morphology, cell proliferation or apoptotic activity. Moreover, the number of metastatic nodules per lung area in nude mice was significantly (P < 0.01) reduced. In conclusion, suppression of Cx43 expression in tumor cells reduced in vitro migration and invasion capacity and in vivo metastatic ability so that Cx43 has potential as a molecular target for prevention of cancer metastasis with Cx43 overexpressing tumors.     

Antagonism of CXCR3 inhibits lung metastasis in a murine model of metastatic breast cancer

Tumor cells aberrantly express chemokines and/or chemokine receptors, and some may promote tumor growth and metastasis. We examined the expression and function of chemokine receptor CXCR3 in a syngeneic murine model of metastatic breast cancer. By flow cytometry, CXCR3 was detected in all murine mammary tumor cell lines examined. All human breast cancer cell lines examined also expressed CXCR3, as did the immortalized but nontumorigenic MCF-10A cell line. Interaction of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, with CXCR3 on the highly malignant murine mammary tumor cell line 66.1 resulted in intracellular calcium mobilization and chemotaxis in vitro. To test the hypothesis that tumor metastasis is facilitated by CXCR3 expressed by tumor cells, we employed a small molecular weight antagonist of CXCR3, AMG487. 66.1 tumor cells were pretreated with AMG487 prior to i.v. injection into immune-competent female mice. Antagonism of CXCR3 on 66.1 tumor cells inhibited experimental lung metastasis, and this antimetastatic activity was compromised in mice depleted of natural killer cells. Systemic administration of AMG487 also inhibited experimental lung metastasis. In contrast to the antimetastatic effect of AMG487, local growth of 66.1 mammary tumors was not affected by receptor antagonism. These studies indicate that murine mammary tumor cells express CXCR3 which facilitates the development of lung metastases. These studies also indicate for the first time that a small molecular weight antagonist of CXCR3 has the potential to inhibit tumor metastasis.     

Curcumin suppresses the paclitaxel-induced nuclear factor-kappaB pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice.

Currently, there is no effective therapy for metastatic breast cancer after surgery, radiation, and chemotherapy have been used against the primary tumor. Because curcumin suppresses nuclear factor-kappaB (NF-kappaB) activation and most chemotherapeutic agents activate NF-kappaB that mediates cell survival, proliferation, invasion, and metastasis, we hypothesized that curcumin would potentiate the effect of chemotherapy in advanced breast cancer and inhibit lung metastasis. We tested this hypothesis using paclitaxel (Taxol)-resistant breast cancer cells and a human breast cancer xenograft model. As examined by electrophoretic mobility gel shift assay, paclitaxel activated NF-kappaB in breast cancer cells and curcumin inhibited it; this inhibition was mediated through inhibition of IkappaBalpha kinase activation and IkappaBalpha phosphorylation and degradation. Curcumin also suppressed the paclitaxel-induced expression of antiapoptotic (XIAP, IAP-1, IAP-2, Bcl-2, and Bcl-xL), proliferative (cyclooxygenase 2, c-Myc, and cyclin D1), and metastatic proteins (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1). It also enhanced apoptosis. In a human breast cancer xenograft model, dietary administration of curcumin significantly decreased the incidence of breast cancer metastasis to the lung and suppressed the expression of NF-kappaB, cyclooxygenase 2, and matrix metalloproteinase-9. Overall, our results indicate that curcumin, which is a pharmacologically safe compound, has a therapeutic potential in preventing breast cancer metastasis possibly through suppression of NF-kappaB and NF-kappaB-regulated gene products.     

Stat3 orchestrates interaction between endothelial and tumor cells and inhibition of Stat3 suppresses brain metastasis of breast cancer cells

Brain metastasis is a major cause of morbidity and mortality in patients with breast cancer. Our previous studies indicated that Stat3 plays an important role in brain metastasis. Here, we present evidence that Stat3 functions at the level of the microenvironment of brain metastases. Stat3 controlled constitutive and inducible VEGFR2 expression in tumor-associated brain endothelial cells. Furthermore, inhibition of Stat3 by WP1066 decreased the incidence of brain metastases and increased survival in a preclinical model of breast cancer brain metastasis. WP1066 inhibited Stat3 activation in tumor-associated endothelial cells, reducing their infiltration and angiogenesis. WP1066 also inhibited breast cancer cell invasion. Our results indicate that WP1066 can inhibit tumor angiogenesis and brain metastasis mediated by Stat3 in endothelial and tumor cells.     

Silencing of STRN4 suppresses the malignant characteristics of cancer cells

The striatin family of proteins, comprising STRN, STRN3 and STRN4, are multidomain‐containing proteins that associate with additional proteins to form a large protein complex. We previously reported that STRN4 directly associated with protein kinases, such as MINK1, TNIK and MAP4K4, which are associated with tumor suppression or tumor progression. However, it remains unclear whether STRN4 is associated with tumor progression. In this report, we examined the role that STRN4 plays in cancer malignancy. We show that depletion of STRN4 suppresses proliferation, migration, invasion and the anchorage‐independent growth of cancer cells. In addition, STRN4 knockdown increases the sensitivity of pancreatic cancer cells to gemcitabine. Finally, we show that STRN4 knockdown suppresses the proliferation and metastasis of cancer cells in mice. Our results demonstrate a possible role of STRN4 in tumor progression.     

Downregulation of CD24 suppresses bone metastasis of lung cancer

Suppression of bone metastasis can improve patient quality of life. Current drugs for bone metastasis have been shown to prolong progression‐free survival but not overall survival; therefore, other potential therapeutic targets for bone metastasis should be investigated. Cell‐surface antigens, such as CD24, have been recently shown to be involved in the metastasis of various cancers. However, whether CD24 plays a role in bone metastasis of lung cancer remains unknown. To observe metastasis of lung cancer cells by imaging technology, we introduced a near‐infrared fluorescent protein, iRFP720, into a bone‐seeking subclone established from lung cancer cells, HARA‐B4 cells. The anchorage‐independent growth of these cells was then evaluated by colony formation assays. We also compared cancer cell tropism to bone tissue with HARA‐B4 cells in the presence or absence of CD24 by cell adhesion assays. To clarify the role of CD24 in bone metastasis, we intracardially injected CD24‐knockdown HARA‐B4 cells into mice and monitored metastasis through detection of iRFP720 using an in vivo imaging system. CD24‐knockdown HARA‐B4 cells in vitro showed reduced anchorage‐independent growth and cancer cell tropism to bone. Bone metastasis was diminished in mice inoculated with CD24‐knockdown HARA‐B4 cells, which was rescued by add‐back of CD24 in cells. Our findings indicate that iRFP720 is effective for in vivo imaging analysis of bone metastasis and that downregulation of CD24 suppresses bone metastasis of lung cancer cells. These findings collectively indicate that CD24 may be considered a promising new therapeutic candidate for the prevention of bone metastasis of lung cancer.     

Reactivation of Syk gene by AZA suppresses metastasis but not proliferation of breast cancer cells

Spleen tyrosine kinase (Syk) is reported to be involved in the suppression of proliferation and invasion of breast cancer. Methylation-mediated Syk gene silencing is found in a subset of breast cancer. In this study, we used a DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (AZA), to restore Syk expression of breast cancer cells. Surprisingly, we found that AZA treatment could reestablish the expression of Syk, but not affect the proliferation of breast cancer cells. Moreover, tumor formation in situ by MDA-MB-435s treated with (+) or without (−) AZA in a nude mice MFP (Mammary fat pad) model did not show significant difference, too. Interestingly, pulmonary metastasis was still significantly suppressed in MDA-MB-435s(+) group (1/9 vs. 7/9). Our findings suggested Syk may be more correlated to metastasis rather than proliferation. This study implied a potential use of Syk methylation as a valuable biomarker to detect high metastatic potential cancerous lesions and the prospect of AZA to join the arsenal of drug candidates to be developed as a new reagent for management of advanced breast cancer.     

MicroRNA-1258 suppresses breast cancer brain metastasis by targeting heparanase

Heparanase (HPSE) is a potent protumorigenic, proangiogenic, and prometastatic enzyme that is overexpressed in brain metastatic breast cancer (BMBC). However, little is known about the regulation of this potential therapeutic target in BMBC, which remains very poorly managed in the clinic. We hypothesized that HPSE gene expression might be regulated by micro RNA that might be exploited therapeutically. Using miRanda and RNAhybrid, we identified miR-1258 as a candidate micro RNA that may directly target HPSE and suppress BMBC. In support of our hypothesis, we found that miR-1258 levels inversely correlated with heparanase expression, enzymatic activity, and cancer cell metastatic propensities, being lowest in highly aggressive BMBC cell variants compared with either nontumorigenic or nonmetastatic human mammary epithelial cells. These findings were validated by analyses of miR-1258 and heparanase content in paired clinical specimens of normal mammary gland versus invasive ductal carcinoma, and primary breast cancer versus BMBC. In regulatory experiments, miR-1258 inhibited the expression and activity of heparanase in BMBC cells, whereas modulating heparanase blocked the phenotypic effects of miR-1258. In functional experiments, stable expression of miR-1258 in BMBC cells inhibited heparanase in vitro cell invasion and experimental brain metastasis. Together, our findings illustrate how micro RNA mechanisms are linked to brain metastatic breast cancer through heparanase control, and they offer a strong rationale to develop heparanase-based therapeutics for treatment of cancer patients with brain metastases, BMBC in particular.     

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC.     

Suppression of lung metastasis of mouse Lewis lung cancer P29 with transfection of the ganglioside GM2/GD2 synthase gene

Ganglioside functions in tumor metastasis were analyzed by carbohydrate remodeling of a mouse Lewis lung cancer (subline P29) by introducing beta1,4GalNAc-T cDNA. Although P29 was originally a low-metastatic subline in the s.c. injection system, it showed high potential in lung metastasis when i.v.-injected via the tail vein. Two lines of GM(2)(+) transfectants showed markedly reduced metastatic potential to the lung compared to 2 control lines. However, cell proliferation rates and expression levels of various cell adhesion molecules, e.g., integrin family members, SLe(x) and CD44, were essentially unchanged after transfection of the cDNA. Then, cell adhesion to fibronectin-coated dishes was examined, showing that GM(2) (+) transfectants attached to the plates much more slowly than controls, suggesting functional modulation of integrins with newly expressed GM(2). Phosphorylation of the FAK located at downstream of integrin molecules was markedly reduced in GM(2)(+) transfectants, suggesting that GM(2) suppressed cell adhesion signals via fibronectin-integrin interaction.