Dorothy Markowitz

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

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.

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.