Yunping Luo

Targeting tumor-associated macrophages as a novel strategy against breast cancer

Tumor-associated macrophages (TAMs) are associated with tumor progression and metastasis. Here, we demonstrate for the first time that legumain, a member of the asparaginyl endopeptidase family functioning as a stress protein, overexpressed by TAMs, provides an ideal target molecule. In fact, a legumain-based DNA vaccine served as a tool to prove this point, as it induced a robust CD8+ T cell response against TAMs, which dramatically reduced their density in tumor tissues and resulted in a marked decrease in proangiogenic factors released by TAMs such as TGF-beta, TNF-alpha, MMP-9, and VEGF. This, in turn, led to a suppression of both tumor angiogenesis and tumor growth and metastasis. Importantly, the success of this strategy was demonstrated in murine models of metastatic breast, colon, and non-small cell lung cancers, where 75% of vaccinated mice survived lethal tumor cell challenges and 62% were completely free of metastases. In conclusion, decreasing the number of TAMs in the tumor stroma effectively altered the tumor microenvironment involved in tumor angiogenesis and progression to markedly suppress tumor growth and metastasis. Gaining better insights into the mechanisms required for an effective intervention in tumor growth and metastasis may ultimately lead to new therapeutic targets and better anticancer strategies.

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.

Downregulation of transcription factor SOX2 in cancer stem cells suppresses growth and metastasis of lung cancer

Background:The cancer stem cell hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells, which have indefinite proliferation and differentiation potentials and give rise to phenotypically diverse cancer cells. Cancer stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the ‘side population’ (SP).Methods and results:The Hoechst efflux assay was used to isolate and characterize the SP from murine D121 lung carcinoma cells. Here, we demonstrated that D121-SP cells contain cancer stem cell characteristics, that is, upregulation of the transcription factors SOX2 and Oct 4 in D121-SP cells. In addition, the migration of D121-SP was decreased, and apoptosis of D121-SP was upregulated following knocking down of SOX2 in D121 cells. Importantly, downregulation of SOX2 in D121 cells markedly suppressed their metastatic potential in syngeneic mice.Conclusions:These results suggest that the SP is an enriched source of lung tumour cells with stem cell properties and that SOX2 has an important role in maintaining stem cell properties and functions that may be a potential target for effective lung cancer therapy.

Tumor‐Associated Macrophages Regulate Murine Breast Cancer Stem Cells Through a Novel Paracrine EGFR/Stat3/Sox‐2 Signaling Pathway

The cancer stem cell (CSC) hypothesis has gained significant recognition as a descriptor of tumorigenesis. Additionally, tumor‐associated macrophages (TAMs) are known to promote growth and metastasis of breast cancer. However, it is not known whether TAMs mediate tumorigenesis through regulation of breast CSCs. Here, we report that TAMs promote CSC‐like phenotypes in murine breast cancer cells by upregulating their expression of Sox‐2. These CSC‐like phenotypes were characterized by increased Sox‐2, Oct‐4, Nanog, AbcG2, and Sca‐1 gene expression, in addition to increased drug‐efflux capacity, resistance to chemotherapy, and increased tumorigenicity in vivo. Downregulation of Sox‐2 in tumor cells by siRNA blocked the ability of TAMs to induce these CSC‐like phenotypes and inhibited tumor growth in vivo. Furthermore, we identified a novel epidermal growth factor receptor (EGFR)/signal transducers and activators of transcription 3 (Stat3)/Sox‐2 paracrine signaling pathway between macrophages and mouse breast cancer cells that is required for macrophage‐induced upregulation of Sox‐2 and CSC phenotypes in tumor cells. We showed that this crosstalk was effectively blocked by the small molecule inhibitors AG1478 or CDDO‐Im against EGFR and Stat3, respectively. Therefore, our report identifies a novel role for TAMs in breast CSC regulation and establishes a rationale for targeting the EGFR/Stat3/Sox‐2 signaling pathway for CSC therapy. STEM CELLS2013;31:248–258

Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine

Protection against breast cancer was achieved with a DNA vaccine against murine transcription factor Fos-related antigen 1, which is overexpressed in aggressively proliferating D2F2 murine breast carcinoma. Growth of primary s.c. tumor and dissemination of pulmonary metastases was markedly suppressed by this oral DNA vaccine, carried by attenuated Salmonella typhimurium, encoding murine Fos-related antigen 1, fused with mutant polyubiquitin, and cotransformed with secretory murine IL-18. The life span of 60% of vaccinated mice was tripled in the absence of detectable tumor growth after lethal tumor cell challenge. Immunological mechanisms involved activation of T, natural killer, and dendritic cells, as indicated by up-regulation of their activation markers and costimulatory molecules. Markedly increased specific target cell lysis was mediated by both MHC class I-restricted CD8+ T cells and natural killer cells isolated from splenocytes of vaccinated mice, including a significant release of proinflammatory cytokines IFN-γ and IL-2. Importantly, fluorescence analysis of fibroblast growth factor 2 and tumor cell-induced vessel growth in Matrigel plugs demonstrated marked suppression of angiogenesis only in vaccinated animals. Taken together, this multifunctional DNA vaccine proved effective in protecting against growth and metastases of breast cancer by combining the action of immune effector cells with suppression of tumor angiogenesis.

Legumain protease-activated TAT-liposome cargo for targeting tumours and their microenvironment

Specific targeting and cellular internalization are key properties for carriers of antitumor therapeutic agents. Here, we develop a drug carrier through the attachment of substrate of endoprotease legumain, alanine-alanine-asparagine (AAN), to cell-penetrating peptides (TAT, trans-activating factor). The addition of the AAN moiety to the fourth lysine in the TAT creates a branched peptide moiety, which leads to a decrease in the transmembrane transport capacity of TAT by 72.65%. Legumain efficiently catalyses the release of TAT-liposome from the AAN-TAT-liposome and thereby recovers the penetrating capacity of TAT. Doxorubicin carried by the AAN-TAT-liposome led to an increase in the tumoricidal effect of doxorubicin and a reduction in its systemic adverse effects in comparison with doxorubicin carried by a control delivery system. Thus, the specific targeting and high efficiency of this delivery platform offers a novel approach to limit the toxicity of anticancer agents as well as increasing their efficacy in cancer therapy.

Oral DNA vaccines target the tumor vasculature and microenvironment and suppress tumor growth and metastasis

Summary: Four novel oral DNA vaccines provide protection against melanoma, colon, breast, and lung carcinoma in mouse models. Vaccines are delivered by attenuated Salmonella typhimurium to secondary lymphoid organs and respectively target vascular endothelial growth factor receptor‐2, transcription factor Fos‐related antigen‐1, anti‐apoptosis protein survivin and Legumain, an asparaginyl endopeptidase specifically overexpressed on tumor‐associated macrophages (TAMs) in the tumor microenvironment (TME). These vaccines are all capable of inducing potent cell‐mediated protective immunity against self‐antigens, resulting in marked suppression of tumor growth and dissemination. Key mechanisms induced by these DNA vaccines include efficient suppression of angiogenesis in the tumor vasculature and marked activation of cytotoxic T cells, natural killer cells, and antigen‐presenting dendritic cells. The vaccine targeting Legumain establishes the new paradigm whereby a reduction in the density of TAMs in the TME decreases the release of factors potentiating tumor growth and angiogenesis. This, in turn, remodels the TME and decreases its immunosuppressive milieu and thereby potentiates the DNA vaccines ability to effectively suppress tumor cell proliferation, vascularization, and metastasis. It is anticipated that such research efforts will lead to novel DNA‐based vaccines that will be effective for the treatment of cancer.

The Role of proto-oncogene Fra-1 in remodeling the tumor microenvironment in support of breast tumor cell invasion and progression

A growing body of evidence indicates that interactions between neoplastic cells and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) are crucial in promoting tumor cell invasion and progression. Macrophages have an ambiguous role in these processes as this M1 phenotype correlates with tumoricidal capacity, whereas TAMs of M2 phenotype exert tumor-promoting effects. In this study, we provide evidence that interactions between mouse breast tumor cells and TAMs remodel the TME, leading to the upregulation of Fra-1, a member of the FOS family of transcription factor. In turn, this proto-oncogene initiates activation of the IL-6/JAK/Stat3 signaling pathway. This creates a malignant switch in breast tumor cells, leading to increased release of proangiogenic factors MMP-9, vascular endothelial growth factor and transforming growth factor-β from tumor cells and intensified invasion and progression of breast cancer. Proof of the concept for the crucial role played by transcription factor Fra-1 in regulating these processes was established by specific knockdown of Fra-1 with small interfering RNA, which resulted in a marked suppression of tumor cell invasion, angiogenesis and metastasis in a mouse breast cancer model. Such a strategy could eventually lead to future efficacious treatments of metastatic breast cancer.

Functional interaction of heat shock protein 90 and Beclin 1 modulates Toll-like receptor-mediated autophagy

Autophagy is one of the downstream effector mechanisms for elimination of intracellular microbes following activation of the Toll‐like receptors (TLRs). Although the detailed molecular mechanism for this cellular process is still unclear, Beclin 1, a key molecule for autophagy, has been suggested to play a role. Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the stability of signaling proteins. Herein, we show that Hsp90 forms a complex with Beclin 1 through an evolutionarily conserved domain to maintain the stability of Beclin 1. In monocytic cells, geldanamycin (GA), an Hsp90 inhibitor, effectively promoted proteasomal degradation of Beclin 1 in a concentration‐dependent (EC50 100 nM) and time‐dependent (t50 2 h) manner. In contrast, KNK437/Hsp inhibitor I had no effect. Hsp90 specifically interacted with Beclin 1 but not with other adapter proteins in the TLR signalsome. Treatment of cells with GA inhibited TLR3‐ and TLR4‐mediated autophagy. In addition, S. typhimurium infection‐induced autophagy was blocked by GA treatment. This further suggested a role of the Hsp90/Beclin 1 in controlling autophagy in response to microbial infections. Taken together, our data revealed that by maintaining the homeostasis of Beclin 1, Hsp90 plays a novel role in TLR‐mediated autophagy.—Xu, C., Liu, J., Hsu, L. ‐C., Luo, Y., Xiang, R., Chuang, T. ‐H. Functional interaction of Hsp90 and Beclin 1 modulates Toll‐like receptor‐mediated autophagy. FASEB J. 25, 2700‐2710 (2011). www.fasebj.org

MicroRNA-19a-3p inhibits breast cancer progression and metastasis by inducing macrophage polarization through downregulated expression of Fra-1 proto-oncogene

One of the hallmarks of malignancy is the polarization of tumor-associated macrophages (TAMs) from a pro-immune (M1-like) phenotype to an immune-suppressive (M2-like) phenotype. However, the molecular basis of the process is still unclear. MicroRNA (miRNA) comprises a group of small, non-coding RNAs that are broadly expressed by a variety of organisms and are involved in cell behaviors such as suppression or promotion of tumorigenesis. Here, we demonstrate that miR-19a-3p, broadly conserved among vertebrates, was downregulated in RAW264.7 macrophage cells of the M2 phenotype in conditoned medium of 4T1 mouse breast tumor cells. This downregulation correlated with an increased expression of the Fra-1 gene, which was reported to act as a pro-oncogene by supporting the invasion and progression of breast tumors. We found significant upregulation of miR-19a-3p in RAW264.7 macrophages after transfection with a miR-19a-3p mimic that resulted in a significant suppression of the expression of this gene. In addition, we could measure the activity of binding between miR-19a-3p and Fra-1 with a psiCHECK luciferase reporter system. Further, transfection of RAW264.7 macrophage cells with the miR-19a-3p mimic decreased the expression of the Fra-1 downstream genes VEGF, STAT3 and pSTAT3. Most importantly, the capacity of 4T1 breast tumor cells to migrate and invade was impaired in vivo by the intratumoral injection of miR-19a-3p. Taken together, these findings indicate that miR-19a-3p is capable of downregulating the M2 phenotype in M2 macrophages and that the low expression of this miRNA has an important role in the upregulation of Fra-1 expression and induction of M2 macrophage polarization.