Supplementary Figure 2 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Legends for Figures 1-6, Table 1 and Methods and Materials from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 6 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 1 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 5 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 3 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Supplementary Figure 4 from The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer

Abstract Purpose: Identification of a generic response biomarker by comparison of chemotherapeutics with different action mechanisms on several noninvasive biomarkers in experimental tumor models. Experimental Design: The spin-lattice relaxation time of water protons (T1) was quantified using an inversion recovery-TrueFISP magnetic resonance imaging method in eight different experimental tumor models before and after treatment at several different time points with five different chemotherapeutics. Effects on T1 were compared with other minimally invasive biomarkers including vascular parameters, apparent diffusion coefficient, and interstitial fluid pressure, and were correlated with efficacy at the endpoint and histologic parameters. Results: In all cases, successful chemotherapy significantly lowered tumor T1 compared with vehicle and the fractional change in T1 (ΔT1) correlated with the eventual change in tumor size (range: r2 = 0.21, P < 0.05 to r2 = 0.73, P < 0.0001), except for models specifically resistant to that drug. In RIF-1 tumors, interstitial fluid pressure was decreased, but apparent diffusion coefficient and permeability increased in response to the microtubule stabilizer patupilone and 5-fluorouracil. Although ΔT1 was small (maximum of −20%), the variability was very low (5%) compared with other magnetic resonance imaging methods (24-48%). Analyses ex vivo showed unchanged necrosis, increased apoptosis, and decreased %Ki67 and total choline, but only Ki67 and choline correlated with ΔT1. Correlation of Ki67 and ΔT1 were observed in other models using patupilone, paclitaxel, a VEGF-R inhibitor, and the mammalian target of rapamycin inhibitor everolimus. Conclusions: These results suggest that a decrease in tumor T1 reflects hypocellularity and is a generic marker of response. The speed and robustness of the method should facilitate its use in clinical trials. Clin Cancer Res; 16(1); 212–25

Noninvasive functional imaging of tumors can provide valuable early-response biomarkers, in particular, for targeted chemotherapy. Using various experimental tumor models, we have investigated the ability of positron emission tomography (PET) measurements of 2-deoxy-2-[(18)F]fluoro-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) to detect response to the allosteric mammalian target of rapamycin (mTOR) inhibitor everolimus. Tumor models were declared sensitive (murine melanoma B16/BL6 and human lung H596) or relatively insensitive (human colon HCT116 and cervical KB31), according to the IC(50) values (concentration inhibiting cell growth by 50%) for inhibition of proliferation in vitro (<10 nM and >1 microM, respectively). Everolimus strongly inhibited growth of the sensitive models in vivo but also significantly inhibited growth of the insensitive models, an effect attributable to its known anti-angiogenic/vascular properties. However, although tumor FDG and FLT uptake was significantly reduced in the sensitive models, it was not affected in the insensitive models, suggesting that endothelial-directed effects could not be detected by these PET tracers. Consistent with this hypothesis, in a well-vascularized orthotopic rat mammary tumor model, other antiangiogenic agents also failed to affect FDG uptake, despite inhibiting tumor growth. In contrast, the cytotoxic patupilone, a microtubule stabilizer, blocked tumor growth, and markedly reduced FDG uptake. These results suggest that FDG/FLT-PET may not be a suitable method for early markers of response to antiangiogenic agents and mTOR inhibitors in which anti-angiogenic/vascular effects predominate because the method could provide false-negative responses. These conclusions warrant clinical testing.

Abstract A limited number of receptor tyrosine kinases (e.g., ErbB and fibroblast growth factor receptor families) have been genetically linked to breast cancer development. Here, we investigated the contribution of the Ret receptor tyrosine kinase to breast tumor biology. Ret was expressed in primary breast tumors and cell lines. In estrogen receptor (ER)α-positive MCF7 and T47D lines, the ligand (glial-derived neurotrophic factor) activated signaling pathways and increased anchorage-independent proliferation in a Ret-dependent manner, showing that Ret signaling is functional in breast tumor cells. Ret expression was induced by estrogens and Ret signaling enhanced estrogen-driven proliferation, highlighting the functional interaction of Ret and ER pathways. Furthermore, Ret was detected in primary cancers, and there were higher Ret levels in ERα-positive tumors. In summary, we showed that Ret is a novel proliferative pathway interacting with ER signaling in vitro. Expression of Ret in primary breast tumors suggests that Ret might be a novel therapeutic target in breast cancer. [Cancer Res 2008;68(10):3743–51]