Promotes Breast Cancer Cell Invasion Research Articles

Tissue resident stem cells produce CCL5 under the influence of cancer cells and thereby promote breast cancer cell invasion

In the present study, we investigated whether human adipose tissue derived stem cells (hASCs) could enhance tumor invasion and whether these hASCs could be a potential source of CCL5. We observed a significant increase in the number of breast cancer cells that invaded the matrigel when Co-cultured with hASCs. We found that hASCs produce CCL5 in the Co-culture and cancer cell invasion was diminished by an antibody against CCL5. Furthermore, cancer cell invasion in the Co-culture was associated with an elevated level of MMP-9 activity. We conclude that CCL5 plays a crucial role for tumor invasion in the interplay of tissue resident stem cells from the fat tissue and breast cancer cells.

Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms

IntroductionThe stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms – one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16).MethodsThe present study has analysed the functional significance of this altered TNC isoform profile in breast cancer. TNC-16 and TNC-14/16 splice variants were generated using PCR-ligation and over-expressed in breast cancer cells (MCF-7, T47D, MDA-MD-231, MDA-MB-468, GI101) and human fibroblasts. The effects of these variants on tumour cell invasion and proliferation were measured and compared with the effects of the large (TNC-L) and fully spliced small (TNC-S) isoforms.ResultsTNC-16 and TNC-14/16 significantly enhanced tumour cell proliferation (P < 0.05) and invasion, both directly (P < 0.01) and as a response to transfected fibroblast expression (P < 0.05) with this effect being dependent on tumour cell interaction with TNC, because TNC-blocking antibodies abrogated these responses. An analysis of 19 matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases 1 to 4 (TIMP 1 to 4) revealed that TNC up-regulated expression of MMP-13 and TIMP-3 two to four fold relative to vector, and invasion was reduced in the presence of MMP inhibitor GM6001. However, this effect was not isoform-specific but was elicited equally by all TNC isoforms.ConclusionsThese results demonstrate a dual requirement for TNC and MMP in enhancing breast cancer cell invasion, and identify a significant role for the tumour-associated TNC-16 and TNC-14/16 in promoting tumour invasion, although these isoform-specific effects appear to be mediated through MMP-independent mechanisms.

G12 signaling through JNK promotes breast cancer cell invasion

The G12 family of heterotrimeric G proteins has been implicated in such cellular processes as cytoskeletal rearrangements, cell‐cell adhesion and oncogenic transformation. Recently, work done in our laboratory has shown that G12 signaling via Rho induces a striking increase in breast cancer cell invasion in vitro and in vivo. In a continuation of this study, we further characterized G12 signaling leading to invasion of breast cancer cells. We show that c‐Jun N‐terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively active Gα12 leads to increased JNK and c‐Jun phosphorylation in the MDA‐MB‐231 breast cancer cell line. Chemical inhibition of JNK or knockdown of JNK by siRNA significantly decreases G12‐induced JNK activation as well as the ability of cells to invade Matrigel in in vitro transwell invasion assays. Furthermore, treatment of these cells with the Rho kinase (ROCK) inhibitor reduces G12‐induced JNK and c‐Jun activation and also G12‐induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. This suggests that a Rho‐ROCK‐JNK signaling axis is stimulated by G12 to promote breast cancer cell invasion. We are currently studying which G protein coupled receptor(s) G12 couples to in order to engage this pathway. This work was supported by a Human Frontier Science Program Fellowship (to J.J.).

Intrinsic genetic characteristics determine tumor-modifying capacity of fibroblasts: matrix metalloproteinase-3 5A/5A genotype enhances breast cancer cell invasion

BackgroundStromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion.MethodsPrimary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided.ResultsTumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04).ConclusionThis study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively.

Angiopoietin-2 Stimulates Breast Cancer Metastasis through the α5β1 Integrin-Mediated Pathway

Acquisition of a metastatic phenotype by breast cancer cells includes alternations of multigenic programs that permit tumor cells to metastasize to distant organs. Here, we report that angiopoietin-2 (Ang2), a known growth factor, is capable of promoting breast cancer cell invasion leading to metastasis. Analysis of 185 primary human breast cancer specimens that include 97 tumors showing lymph node and/or distant metastasis reveals a significant correlation between the expression of Ang2 and E-cadherin, Snail, metastatic potential, tumor grade, and lymph-vascular invasion during breast cancer progression. Using a xenograft model, we show that overexpression of Ang2 in poorly metastatic MCF-7 breast cancer cells suppresses expression of E-cadherin and induces Snail expression and phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) promoting metastasis to the lymph nodes and lung. In cell culture, Ang2 promotes cell migration and invasion in Tie2-deficient breast cancer cells through the alpha(5)beta(1) integrin/integrin-linked kinase (ILK)/Akt, GSK-3beta/Snail/E-cadherin signaling pathway. Inhibition of ILK and the alpha(5)beta(1) integrin abrogates Ang2 modulation of Akt, GSK-3beta, Snail, and E-cadherin and Ang2-stimulated breast cancer cell migration and invasion. Together, these results underscore the significant contribution of Ang2 in cancer progression, not only by stimulating angiogenesis but also by promoting metastasis, and provide a mechanism by which breast cancer cells acquire an enhanced invasive phenotype contributing to metastasis.

D4-GDI, a Rho GTPase Regulator, Promotes Breast Cancer Cell Invasiveness

D4-GDI is a Rho GDP dissociation inhibitor that is widely expressed in hematopoietic cells. Its possible expression and function in breast cancer cells has not been described. Here, we found that D4-GDI is expressed in a panel of breast cancer cell lines, but not in benign-derived mammary epithelial cells. Knockdown of D4-GDI expression in MDA-MB-231 cells by RNA interference blocks cell motility and invasion. The cells lacking D4-GDI grown on Matrigel revert to a normal breast epithelial phenotype characterized by the formation of cavitary structures. Silencing D4-GDI expression inhibits beta1-integrin expression and cell-matrix adhesion. Reintroduction of D4-GDI fully restored both beta1-integrin expression and cellular invasion. Knockdown of D4-GDI in BT549 cells results in a similar effect. These results show that D4-GDI modulates breast cancer cell invasive activities.

Activation of Abl Tyrosine Kinases Promotes Invasion of Aggressive Breast Cancer Cells

The Abl family of nonreceptor tyrosine kinases consists of two related proteins, c-Abl and Abl-related gene (Arg). Activated forms of the Abl kinases (BCR-Abl, Tel-Abl, and Tel-Arg) induce the development of human leukemia; it is not known, however, whether Abl kinases are activated in solid tumors or whether they contribute to tumor development or progression. Previously, we showed that Abl kinases are activated downstream of growth factor receptors, Src family kinases, and phospholipase Cgamma1 (PLCgamma1) in fibroblasts and influence growth factor-mediated proliferation, membrane ruffling, and migration. Growth factor receptors, Src kinases, and PLCgamma1 are deregulated in many solid tumors and drive tumor invasion and metastasis. In this study, we found that Abl kinases are constitutively activated, in highly invasive breast cancer cell lines, downstream of deregulated ErbB receptors and Src kinases. Furthermore, activation of Abl kinases promotes breast cancer cell invasion, as treatment of cells with the Abl kinase inhibitor, STI571, or silencing c-Abl and Arg expression with RNA interference dramatically inhibits Matrigel invasion. This is the first evidence that (a) Abl kinases are deregulated and activated in a nonhematopoietic cancer, (b) activation of Abl kinases in breast cancer cells occurs via a novel mechanism, and (c) constitutive activation of Abl kinases promotes invasion of breast cancer cells. These data suggest that pharmacologic inhibitors targeted against Abl kinases could potentially be useful in preventing breast cancer progression in tumors harboring activated Abl kinases.

The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis

Although the prognosis for patients with early-stage breast cancer has improved, the therapeutic options for patients with locally advanced and metastatic disease are limited. To improve the treatment of these patients, the molecular mechanisms underlying breast cancer invasion and metastasis must be understood. In this study, we report that signaling through the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) promotes breast cancer cell invasion. Moreover, we demonstrate that inhibition of G12 signaling reduces the metastatic dissemination of breast cancer cells in vivo. Finally, we demonstrate that the expression of Galpha12 is significantly up-regulated in the earliest stages of breast cancer, implying that amplification of G12 signaling may be an early event in breast cancer progression. Taken together, these observations identify the G12 family proteins as important regulators of breast cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced patient morbidity and mortality.

NFAT Induces Breast Cancer Cell Invasion by Promoting the Induction of Cyclooxygenase-2

The NFAT (nuclear factor of activated T cells) family of transcription factors plays a fundamental role in the transcriptional regulation of the immune response. However, NFATs are ubiquitously expressed, and recent evidence points to their important functions in human epithelial cells and carcinomas. Specifically, NFAT has been shown to be active in human breast and colon carcinoma cells and to promote their invasion through Matrigel. The mechanisms by which NFAT promotes invasion have not been defined. To identify NFAT target genes that induce carcinoma invasion, we have established stable breast cancer cell lines that inducibly express transcriptionally active NFAT. Gene expression profiling by cDNA microarray of cells induced to express NFAT revealed up-regulation of cyclooxygenase-2 (COX-2). Increased NFAT expression and activity induced COX-2 expression as well as prostaglandin E2 synthesis. This induction was more prominent when NFAT was activated by phorbol 12-myristate 13-acetate and calcium ionophore ionomycin and was blocked by the NFAT antagonist cyclosporin A. Breast cancer cells with elevated COX-2 expression showed increased invasion through Matrigel, and this was reduced in cells treated with COX-2 inhibitors. Conversely, loss of NFAT1 protein expression using small interfering RNA led to a reduction in COX-2 transcription and reduced invasion. Similarly, Matrigel invasion was reduced in cells in which COX-2 expression was reduced using specific siRNA. These findings demonstrate that NFAT promotes breast cancer cell invasion through the induction of COX-2 and the synthesis of prostaglandins.

Upregulation and activation of PKCα by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKCα and Src inhibitors

Although ErbB2 is known to enhance breast cancer metastasis, the signaling events responsible for this remain elusive. Alpha-isozyme of protein kinase C (PKCalpha), which is involved in cancer development and progression, has been suggested to be activated by ErbB2 without direct evidence. In addition, the roles of PKCalpha in ErbB2-mediated cancer cell malignancy have not been clearly identified. In this study, we investigated whether ErbB2 can activate PKCalpha and determined what role PKCalpha plays in ErbB2-mediated breast cancer cell invasion. We expressed wild-type and mutant ErbB2 with altered signaling capacities in MDA-MB-435 breast cancer cells and revealed that overexpression or activation of ErbB2 in MDA-MB-435 cells upregulated and activated PKCalpha and that downregulation of ErbB2 by small-interfering RNA decreased the expression and activity of PKCalpha in BT474 breast cancer cells. These in vitro results were supported by data from breast cancer patient samples. In 150 breast cancer tumor samples, ErbB2-overexpressing tumors showed significantly higher positive rates of PKCalpha membrane immunohistochemistry staining than that of ErbB2-low-expressing tumors. Mechanistically, we found that PKCalpha is co-immunoprecipitated with Src and PKCalpha expression and activity can be decreased by Src inhibitor PP2 and by the expression of a dominant-negative mutant of Src. Moreover, ErbB2-mediated upregulation of urokinase-type plasminogen activator receptor (uPAR) is reduced by either the PKCalpha inhibitor Go6976 or the Src inhibitor PP2, and the combination of Go6976 with PP2 is superior to either agent alone in suppressing uPAR expression and cell invasion. These results demonstrate that PKCalpha is critical for ErbB2-mediated cancer cell invasion and provide valuable insights for current and future PKCalpha and Src inhibitor clinical trials.

Fibroblasts promote breast cancer cell invasion by upregulating tumor matrix metalloproteinase–9 production

Background. Thrombospondin-1 (TSP-1) promotes breast cancer cell invasion of collagen by upregulating matrix metalloproteinase–9 (MMP-9) production. Stromal TSP-1 may play a role in regulating tumor cell invasion. We hypothesize that fibroblasts promote breast cancer cell invasion by upregulating the production of MMP-9 through TSP-1. Methods. MDA-MB-231 human breast carcinoma cells were grown alone or in coculture with human fibroblasts. Gelatin zymography and Western immunoblot analysis for MMP-9 were performed on the coculture cell media and the single cell media. Inhibition of fibroblast-mediated breast tumor cell invasion by an anti-TSP-1 or an anti-MMP-9 antibody was evaluated using a modified Boyden chamber. Results. Coculture experiments showed an increased production of MMP-9 when compared with breast cancer single cell culture or fibroblast single cell culture experiments as demonstrated by zymography and Western immunoblot analysis. Fibroblast-stimulated MMP-9 production was comparable with TSP-1-stimulated MMP-9 production. Anti-TSP-1 antibody and anti-MMP-9 antibody inhibited fibroblast-stimulated tumor cell invasion to 30% and 26% of controls, respectively (P <.05). Conclusions. Fibroblasts may regulate breast cancer cell invasion by promoting tumor MMP-9 production through TSP-1. Inhibition of stromal TSP-1 stimulation of MMP-9 synthesis may prevent matrix degradation necessary for tumor invasion and metastasis. (Surgery 2002;132:220-5.)