Circulating SOD2 Is a Candidate Response Biomarker for Neoadjuvant Therapy in Breast Cancer.

There is a great need for non-invasive tools that inform of an early molecular response to cancer therapeutic treatment. Here, we tested the hypothesis that proteolytically resistant proteins could be candidate circulating tumor biomarkers for cancer therapy. Proteins resistant to proteolysis are drastically under-sampled by current proteomic workflows. These proteins could be reliable sensors for the response to therapy since they are likely to stay longer in circulation. We selected manganese superoxide dismutase (SOD2), a mitochondrial redox enzyme, from a screening of proteolytic resistant proteins in breast cancer (BC). First, we confirmed the robustness of SOD2 and determined that its proteolytic resistance is mediated by its quaternary protein structure. We also proved that the release of SOD2 upon chemotherapy treatment correlates with cell death in BC cells. Then, after confirming that SOD2 is very stable in human serum, we sought to measure its circulating levels in a cohort of BC patients undergoing neoadjuvant therapy. The results showed that circulating levels of SOD2 increased when patients responded to the treatment according to the tumor shrinkage during neoadjuvant chemotherapy. Therefore, the measurement of SOD2 levels in plasma could improve the non-invasive monitoring of the therapeutic treatment in breast cancer patients. The identification of circulating biomarkers linked to the tumor cell death induced by treatment could be useful for monitoring the action of the large number of cancer drugs currently used in clinics. We envision that our approach could help uncover candidate tumor biomarkers to measure a tumor's response to cancer therapy in real time by sampling the tumor throughout the course of treatment.

V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy.

In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.

ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines.

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3ß blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

E2F1 regulates cellular growth by mTORC1 signaling.

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.