Search

Experimental treatments (A) and tumor size follow-up until sacrifice (B) of tumors used to prepare cell suspensions for figures 3, 5 and supplementary figure 4. (C) Shows individual tumor lesion weights used to calculate lymphocytes/gram densities.

FACS analyses on cell suspensions (n=4) derived from non-irradiated MC38-derived tumors

Myeloid cell content (density per gram) in cell suspension from non-irradiated tumors as those in figure 5. Macrophages are defined as CD45+ CD11b+ F4/80+; Dendritic cells (DC) as CD45+CD11b+CD11c+; myeloid-derived suppressor cells (MDSC) as CD45+CD11b+Ly6C+ or CD45+CD11b+Ly6G+ to estimate monocytic (M-MDSC) and granulocytic (G-MDSC) subtypes.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti–PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGFβ expression or activation increases in irradiated tissues, we tested whether TGFβ blockade may further enhance abscopal effects in conjunction with the anti–PD-1 plus anti-CD137 mAb combination. Indeed, TGFβ blockade with 1D11, a TGFβ-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGFβ in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGFβ hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGFβ blockade in combination with radioimmunotherapy results in greater efficacy.

Assessment of spontaneous lung metastases of 4T1 tumors in the indicated treatment groups corresponding to mice treated as in figure 2A. Metastases were detected and quantitated -number of lesions in the surface of the lung counted upon inspection under a magnification lens (left) and by real time RT-PCR for tumor specific gp70 RNA (right)-.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti–PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGFβ expression or activation increases in irradiated tissues, we tested whether TGFβ blockade may further enhance abscopal effects in conjunction with the anti–PD-1 plus anti-CD137 mAb combination. Indeed, TGFβ blockade with 1D11, a TGFβ-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGFβ in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGFβ hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGFβ blockade in combination with radioimmunotherapy results in greater efficacy.

eIF4E is frequently overexpressed in tumors of the breast, colon, prostate, and lung, and expression correlates with disease progression, increased tissue invasion and metastasis. It has been suggested that overexpression of eIF4E contributes to malignancy by selectively increasing translation of mRNAs containing significant secondary structure in the their 5' noncoding regions. A limited group of mRNAs encode key proteins involved in cellular growth, survival, angiogenesis and malignancy and contain long GC-rich highly structured 5' noncoding regions. The effect of increased levels of eIF4E in tumor cells, however, is still not well understood, nor is the role of its mTOR regulated repressor, 4E-BP1. Here we demonstrate the surprising finding that overexpression of eIF4E acts primarily to increase breast cancer cell invasion by significantly increasing translation of a single mRNA: β1 integrin. eIF4E overexpression at levels found in breast cancers (3-4 fold) results in a 10-fold increased level of tumor cell invasion. Analysis of polysomal mRNA found increased translation of β1 integrin mRNA with physiological levels of eIF4E overexpression observed in breast cancers. We found that increased β1 integrin expression was due to increased read-through of the complex GC-rich 5' noncoding region of β1 integrin mRNA following eIF4E overexpression, the first time this has been described physiologically, and that this effect is blocked by co-overexpression of 4E-BP1. The modest increase in β1 integrin protein levels is amplified by a disproportionately large increase in the formation of the cell surface heterodimer α3β1, an integrin frequently implicated in breast tumor invasion. These and other results can account for the large increase in invasive ability in eIF4E overexpressing breast cancer cells, suggesting that β1 integrin levels may be limiting in the cell. In addition, we found that increased expression of eIF4E results in increased transcription and consequent protein levels of many canonical TGFβ-responsive genes, including β1 integrin. TGFβ is partially responsible for the increased invasion seen with eIF4E overexpression, although cells are still 4.5 fold more invasive even in the absence of the cytokine, consistent with the TGFβ-independent increase in β1 integrin levels observed with eIF4E overexpresison. The TGFβ transcriptional effect is likely due to an increased rate of activation of latent TGFβ (typically present in serum or the stroma), mediated by the eIF4E-driven increased levels of β1 integrin and its subsequent heterodimerization with α integrins capable of activating TGFβ, including αvβ1. TGFβ transcriptional effects are due to TGFβ ligation with its receptor, and not eIF4E-mediated increased translation of a downstream TGFβ effector, as they are blocked with the use of a TGFβ receptor I kinase inhibitor. Additionally, eIF4E overexpression does not facilitate the translation of TGFβ mRNA, as protein levels of TGFβ are not increased with elevated levels of eIF4E. Co-overexpression of 4E-BP1 blocks all TGFβ effects. Our results suggest a new but prevalent model whereby eIF4E level is a master regulator that determines the direction of TGFβ activity. Increased expression of eIF4E at typical human breast cancer levels mediates increased tumor cell invasion through the selective increased translation of β1 integrin mRNA. Higher levels of β1 integrin promotes increased heterodimerization with α-integrins which activate latent TGFβ present in the breast stroma, thereby directing TGFβ to pro-invasive function. Citation Format: Celine Mestel, Mary-Helen Barcelos-Hoff, Jiri Zavadil, Silvia C. Formenti, Robert J. Schneider. eIF4E in breast cancer is a master regulator of tumor cell invasion through increased translation of beta-1 integrin mRNA and TGF-beta activation [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY35-03