Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin

BackgroundBreast cancer is a leading cause of death in women and with an increasing worldwide incidence. Doxorubicin, as a first-line anthracycline-based drug is conventional used on breast cancer clinical chemotherapy. However, the drug resistances limited the curative effect of the doxorubicin therapy in breast cancer patients, but the molecular mechanism determinants of breast cancer resistance to doxorubicin chemotherapy are not fully understood. In order to explore the association between metadherin (MTDH) and doxorubicin sensitivity, the differential expressions of MTDH in breast cancer cell lines and the sensitivity to doxorubicin of breast cancer cell lines were investigated.MethodsThe mRNA and protein expression of MTDH were determined by real-time PCR and Western blot in breast cancer cells such as MDA-MB-231, MCF-7, MDA-MB-435S, MCF-7/ADR cells. Once MTDH gene was knocked down by siRNA in MCF-7/ADR cells and overexpressed by MTDH plasmid transfection in MDA-MB-231 cells, the cell growth and therapeutic sensitivity of doxorubicin were evaluated using MTT and the Cell cycle assay and apoptosis rate was determined by flow cytometry.ResultsMCF-7/ADR cells revealed highly expressed MTDH and MDA-MB-231 cells had the lowest expression of MTDH. After MTDH gene was knocked down, the cell proliferation was inhibited, and the inhibitory rate of cell growth and apoptosis rate were enhanced, and the cell cycle arrest during the G0/G1 phase in the presence of doxorubicin treatment. On the other hand, the opposite results were observed in MDA-MB-231 cells with overexpressed MTDH gene.ConclusionMTDH gene plays a promoting role in the proliferation of breast cancer cells and its high expression may be associated with doxorubicin sensitivity of breast cancer.     

Metadherin mediates lipopolysaccharide-induced migration and invasion of breast cancer cells

Background

Breast cancer is the most prevalent cancer in women worldwide and metastatic breast cancer has very poor prognosis. Inflammation has been implicated in migration and metastasis of breast cancer, although the exact molecular mechanism remains elusive.

Principal Findings

We show that the pro-inflammatory endotoxin Lipopolysaccharide (LPS) upregulates the expression of Metadherin (MTDH), a recently identified oncogene, in a number of breast cancer lines. Stable knockdown of MTDH by shRNA in human breast MDA-MB-231 cells abolishes LPS-induced cell migration and invasion as determined by several in vitro assays. In addition, knockdown of MTDH diminishes Nuclear Factor-kappa B (NF-κB) activation by LPS and inhibited LPS-induced IL-8 and MMP-9 production.

Conclusions

These results strongly suggest that MTDH is a pivotal molecule in inflammation-mediated tumor metastasis. Since NF-κB, IL-8 and MMP-9 play roles in LPS-induced invasion or metastasis, the mechanism of MTDH-promoted invasion and metastasis may be through the activation of NF-κB, IL-8 and MMP-9, also suggesting a role of MTDH in regulating both inflammatory responses and inflammation-associated tumor invasion. These findings indicate that MTDH is involved in inflammation-induced tumor progression, and support that MTDH targeting therapy may hold promising prospects in treating breast cancer.     

Identification of novel variants of metadherin in breast cancer

Metadherin (MTDH, also known as AEG-1, and Lyric) has been demonstrated to play a potential role in several significant aspects of tumor progression. It has been reported that overexpression of MTDH is associated with progression of disease and poorer prognosis in breast cancer. However, there are no studies to date assessing variants of the MTDH gene and their potential relationship with breast cancer susceptibility. Thus, we investigated all variants of the MTDH gene and explored the association of the variants with breast cancer development. Our cohort consisted of full-length gene sequencing of 108 breast cancer cases and 100 healthy controls; variants were detected in 11 breast cancer cases and 13 controls. Among the variants detected, 9 novel variants were discovered and 2 were found to be associated with the susceptibility of breast cancer. However, additional studies need to be conducted in larger sample sizes to validate these findings and to further investigate whether these variants are prognostic in breast cancer patients.     

Identification of staphylococcal nuclease domain-containing 1 (SND1) as a Metadherin-interacting protein with metastasis-promoting functions

Metastasis is the deadliest and most poorly understood feature of malignant diseases. Recent work has shown that Metadherin (MTDH) is overexpressed in over 40% of breast cancer patients and promotes metastasis and chemoresistance in experimental models of breast cancer progression. Here we applied mass spectrometry-based screen to identify staphylococcal nuclease domain-containing 1 (SND1) as a candidate MTDH-interacting protein. After confirming the interaction between SND1 and MTDH, we tested the role of SND1 in breast cancer and found that it strongly promotes lung metastasis. SND1 was further shown to promote resistance to apoptosis and to regulate the expression of genes associated with metastasis and chemoresistance. Analyses of breast cancer clinical microarray data indicated that high expression of SND1 in primary tumors is strongly associated with reduced metastasis-free survival in multiple large scale data sets. Thus, we have uncovered SND1 as a novel MTDH-interacting protein and shown that it is a functionally and clinically significant mediator of metastasis.     

A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis. The suppression of TAMs in the tumor-microenvironment (TME) provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor’s stroma. Here, we tested our hypothesis that suppression of TAMs can be achieved in syngeneic BALB/c mice with oral minigene vaccines against murine MHC class I antigen epitopes of Legumain, an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which is overexpressed on TAMs in the tumor stroma. Vaccine vectors were constructed and transformed into attenuated Salmonella typhimurium (Dam ⁻ , AroA ⁻ ) for oral delivery. Groups of mice received either the expression vectors encoding the Legumain H-2D or 2K epitopes or the control empty vector by gavage. The efficacy of the minigene vaccines was determined by their ability to protect mice from lethal tumor cell challenges, the induction of a specific CTL response as well as IFN-γ release, and inhibition of tumor angiogenesis. We demonstrated that the Legumain minigene vaccine provided effective protection against tumor cell challenge by inducing a specific CD8⁺ T-cell response against Legumain⁺ TAMs in our breast tumor model. The protection, induced by this T-cell response, mediated by the Legumain Kd minigene, is also responsible for lysing D2F2 breast carcinoma cells in syngeneic BALB/c mice and for suppressing tumor angiogenesis. Importantly, in a prophylactic setting, the minigene vaccine proved to be of similar anti-tumor efficacy as a vaccine encoding the entire Legumain gene. Together, our findings establish proof of concept that a Legumain minigene vaccine provides a more flexible alternative to the whole gene vaccine, which may facilitate the future design and clinical applications of such a vaccine for cancer prevention.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

Suberoylanilide Hydroxamic Acid,an Inhibitor of Histone Deacetylase,Enhances Radiosensitivity and Suppresses Lung Metastasis in Breast Cancer In Vitro and In Vivo

Triple-negative breast cancer (TNBC), defined by the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, is associated with an early recurrence of disease and poor outcome. Furthermore, the majority of deaths in breast cancer patients are from metastases instead of from primary tumors. In this study, MCF-7 (an estrogen receptor-positive human breast cancer cell line), MDA-MB-231 (a human TNBC cell line) and 4T1 (a mouse TNBC cell line) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with suberoylanilide hydroxamic acid (SAHA, an inhibitor of histone deacetylase (HDAC)) and to determine the underlying mechanisms of these effects in vitro and in vivo. We also evaluated the ability of SAHA to inhibit the metastasis of 4T1 cells. We found that IR combined with SAHA showed increased therapeutic efficacy when compared with either treatment alone in MCF-7, MDA-MB-231 and 4T1 cells. Moreover, the combined treatment enhanced DNA damage through the inhibition of DNA repair proteins. The combined treatment was induced primarily through autophagy and ER stress. In an orthotopic breast cancer mouse model, the combination treatment showed a greater inhibition of tumor growth. In addition, SAHA inhibited the migration and invasion abilities of 4T1 cells and inhibited breast cancer cell migration by inhibiting the activity of MMP-9. In an in vivo experimental metastasis mouse model, SAHA significantly inhibited lung metastasis. SAHA not only enhances radiosensitivity but also suppresses lung metastasis in breast cancer. These novel findings suggest that SAHA alone or combined with IR could serve as a potential therapeutic strategy for breast cancer.     

Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity

Background

Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer.

Methodology/Principal Findings

Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8⁺ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect.

Conclusions/Significance

Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer immune-based therapy.     

Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2^∗, -193b and -193a, and inversely inhibit miR-31 and -9^∗. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.     

BRCC2 inhibits breast cancer cell growth and metastasis in vitro and in vivo via downregulating AKT pathway

In our previous study, we demonstrated that the BRCC2 (breast cancer cell 2) gene is a proapoptotic molecule that interacts with Bcl-X_L. BRCC2 downregulation is associated with poor disease-free and overall survival in breast cancer. In this study, we aimed to investigate the role of BRCC2 in tumor suppression in breast cancer. In clinical breast cancer samples, we found that BRCC2 expression was significantly downregulated in cancer lesions compared with paired normal breast tissues. By silencing or overexpressing BRCC2 in breast cancer cells, we found that BRCC2 could inhibit cell growth and metastasis in vitro. An in vivo assay showed that BRCC2 not only dramatically inhibited breast cancer cell xenograft formation and growth but also inhibited breast cancer cell metastasis in a lung metastasis model. Moreover, we demonstrated that BRCC2 inhibited breast cancer metastasis via regulation of the Akt pathway. Thus, our study provided evidence that BRCC2 functions as a novel tumor suppressor in breast cancer and may be a potential therapeutic target for breast cancer management.     

Schisandrin B attenuates cancer invasion and metastasis via inhibiting epithelial-mesenchymal transition

Background

Metastasis is the major cause of cancer related death and targeting the process of metastasis has been proposed as a strategy to combat cancer. Therefore, to develop candidate drugs that target the process of metastasis is very important. In the preliminary studies, we found that schisandrin B (Sch B), a naturally-occurring dibenzocyclooctadiene lignan with very low toxicity, could suppress cancer metastasis.

Methodology

BALB/c mice were inoculated subcutaneously or injected via tail vein with murine breast cancer 4T1 cells. Mice were divided into Sch B-treated and control groups. The primary tumor growth, local invasion, lung and bone metastasis, and survival time were monitored. Tumor biopsies were examined immuno- and histo-pathologically. The inhibitory activity of Sch B on TGF-β induced epithelial-mesenchymal transition (EMT) of 4T1 and primary human breast cancer cells was assayed.

Principal Findings

Sch B significantly suppressed the spontaneous lung and bone metastasis of 4T1 cells inoculated s.c. without significant effect on primary tumor growth and significantly extended the survival time of these mice. Sch B did not inhibit lung metastasis of 4T1 cells that were injected via tail vein. Delayed start of treatment with Sch B in mice with pre-existing tumors did not reduce lung metastasis. These results suggested that Sch B acted at the step of local invasion. Histopathological evidences demonstrated that the primary tumors in Sch B group were significantly less locally invasive than control tumors. In vitro assays demonstrated that Sch B could inhibit TGF-β induced EMT of 4T1 cells and of primary human breast cancer cells.

Conclusions

Sch B significantly suppresses the lung and bone metastasis of 4T1 cells via inhibiting EMT, suggesting its potential application in targeting the process of cancer metastasis.     

Active immunization with a Her-2/neu-targeting Multi-peptide B cell vaccine prevents lung metastases formation from Her-2/neu breast cancer in a mouse model

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4⁺ and CD8⁺ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer.     

Akt1 inhibition promotes breast cancer metastasis through EGFR-mediated β-catenin nuclear accumulation

Background

Knockdown of Akt1 promotes Epithelial-to-Mesenchymal Transition in breast cancer cells. However, the mechanisms are not completely understood.

Methods

Western blotting, immunofluorescence, luciferase assay, real time PCR, ELISA and Matrigel invasion assay were used to investigate how Akt1 inhibition promotes breast cancer cell invasion in vitro. Mouse model of lung metastasis was used to measure in vivo efficacy of Akt inhibitor MK2206 and its combination with Gefitinib.

Results

Knockdown of Akt1 stimulated β-catenin nuclear accumulation, resulting in breast cancer cell invasion. β-catenin nuclear accumulation induced by Akt1 inhibition depended on the prolonged activation of EGFR signaling pathway in breast cancer cells. Mechanistic experiments documented that knockdown of Akt1 inactivates PIKfyve via dephosphorylating of PIKfyve at Ser³¹⁸ site, resulting in a decreased degradation of EGFR signaling pathway. Inhibition of Akt1 using MK2206 could induce an increase in the expression of EGFR and β-catenin in breast cancer cells. In addition, MK2206 at a low dosage enhance breast cancer metastasis in a mouse model of lung metastasis, while an inhibitor of EGFR tyrosine kinase Gefitinib could potentially suppress breast cancer metastasis induced by Akt1 inhibition.

Conclusion

EGFR-mediated β-catenin nuclear accumulation is critical for Akt1 inhibition-induced breast cancer metastasis.

     

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model

Background

Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration.

Methods

In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine.

Results

The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicty T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunityas indicated by depletion of lymphocytes in vivo.

Conclusion

Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.     

The pattern of clinical breast cancer metastasis correlates with a single nucleotide polymorphism in the C1qA component of complement

Complement is one of primary defense mechanisms against intravascular microorganisms and could play a role in the immune response to malignancy and hence its clinical behavior. We evaluated if the sole coding polymorphism of C1qA associates with outcome in patients with breast carcinoma. Genotyping for C1qA_[276A/G] was performed in 63 breast cancer subjects with localized tumor and compared with that in 38 breast cancer subjects with metastasis. Established risk factors for clinical outcome were considered and evaluated in multivariable analysis. Breast cancer subjects with heterozygous or homozygous C1qA_[276G] genotype had a higher rate of metastasis than subjects with the homozygous C1qA_[276A] genotype [hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.1–4.1]. This association was stronger when only metastatic sites associated with hematogenous spread, i.e., to the bone, liver, and brain, were considered (HR 3.5, 95% CI 1.4–5.6) and remained statistically significant after adjustment for the number of positive lymph nodes, estrogen receptor status, and progesterone receptor status. There was no statistical difference in the C1qA_[276A/G] allelic distribution between all subjects with breast cancer and controls. These results suggest there could be an association of a single nucleotide polymorphism at position 276 of the C1qA component of complement with breast cancer metastasis to sites linked to hematogenous spread of disease. The C1qA polymorphism associated with decreased distant metastasis has also been correlated with an increased incidence of subcutaneous systemic lupus and C1q deficiencies, suggesting that an altered immune response may play a role in the observed association. Supported in part by National Institute of Health grant R21-CA90822, Friends You Can Count On! grant 1-87093-00, and the Woody and Louise White Cancer Research Fund.     

Intraperitoneal Administration of a Tumor-Associated Antigen SART3, CD40L,and GM-CSF Gene-Loaded Polyplex Micelle Elicits a Vaccine Effect in Mouse Tumor Models

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c⁺ DCs and CD4⁺/CD8a⁺ T cells into tumors. Depletion of CD4⁺ or CD8a⁺ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Alginic Acid-Coated Chitosan Nanoparticles Loaded with Legumain DNA Vaccine: Effect against Breast Cancer in Mice

Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl₂, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3⁺/CD8⁺/CD25⁺) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.