Glioblastoma endothelium drives bevacizumab-induced infiltrative growth via modulation of PLXDC1

Bevacizumab, a VEGF-targeting monoclonal antibody, may trigger an infiltrative growth pattern in glioblastoma. We investigated this pattern using both a human specimen and rat models. In the human specimen, a substantial fraction of infiltrating tumor cells were located along perivascular spaces in close relationship with endothelial cells. Brain xenografts of U87MG cells treated with bevacizumab were smaller than controls (p = 0.0055; Student t-test), however, bands of tumor cells spread through the brain farther than controls (p < 0.001; Student t-test). Infiltrating tumor Cells exhibited tropism for vascular structures and propensity to form tubules and niches with endothelial cells. Molecularly, bevacizumab triggered an epithelial to mesenchymal transition with over-expression of the receptor Plexin Domain Containing 1 (PLXDC1). These results were validated using brain xenografts of patient-derived glioma stem-like cells. Enforced expression of PLXDC1 in U87MG cells promoted brain infiltration along perivascular spaces. Importantly, PLXDC1 inhibition prevented perivascular infiltration and significantly increased the survival of bevacizumab-treated rats. Our study indicates that bevacizumab-induced brain infiltration is driven by vascular endothelium and depends on PLXDC1 activation of tumor cells

A method to assess target gene involvement in angiogenesis in vitro and in vivo using lentiviral vectors expressing shRNA.

Current methods to study angiogenesis in cancer growth and development can be difficult and costly, requiring extensive use of in vivo methodologies. Here, we utilized an in vitro adipocyte derived stem cell and endothelial colony forming cell (ADSC/ECFC) co-culture system to investigate the effect of lentiviral-driven shRNA knockdown of target genes compared to a non-targeting shRNA control on cord formation using High Content Imaging. Cord formation was significantly reduced following knockdown of the VEGF receptor VEGFR2 in VEGF-driven cord formation and the FGF receptor FGFR1 in basic FGF (bFGF)-driven cord formation. In addition, cord formation was significantly reduced following knockdown of the transcription factor forkhead box protein O1 (FOXO1), a protein with known positive effects on angiogenesis and blood vessel stabilization in VEGF- and bFGF-driven cord formation. Lentiviral shRNA also demonstrated utility for stable knockdown of VEGFR2 and FOXO1 in ECFCs, allowing for interrogation of protein knockdown effects on in vivo neoangiogenesis in a Matrigel plug assay. In addition to interrogating the effect of gene knockdown in endothelial cells, we utilized lentiviral shRNA to knockdown specificity protein 1 (SP1), a transcription factor involved in the expression of VEGF, in U-87 MG tumor cells to demonstrate the ability to analyze angiogenesis in vitro in a tumor-driven transwell cord formation system and in tumor angiogenesis in vivo. A significant reduction in tumor-driven cord formation, VEGF secretion, and in vivo tumor angiogenesis was observed upon SP1 knockdown. Therefore, evaluation of target gene knockdown effects in the in vitro co-culture cord formation assay in the ADSC/ECFC co-culture, ECFCs alone, and in tumor cells translated directly to in vivo results, indicating the in vitro method as a robust, cost-effective and efficient in vitro surrogate assay to investigate target gene involvement in endothelial or tumor cell function in angiogenesis

Induction of SESN3 mRNA expression by ponatinib in T315I-BCR-ABL expressing cells.

<p><b>A</b>. Ba/F3 cells stably transfected with T315I-BCR-ABL were treated with ponatinib (10 nM) or imatinib (1 µM) for 12 hours as indicated. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using β-actin for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>B</b>. BV173R cells were treated with ponatinib (100 nM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 4 independent experiments.</p

Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells

<div><p>Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.</p></div

Inhibitory effects of SESN3 but not SESN2 on primitive BCR-ABL expressing leukemic progenitors.

<p><b>A</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or a SESN3 expressing plasmid. Levels of SESN3 were quantified at 24 hours post-nucleofection by immunoblotting. <b>B</b>. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN3 were plated in methylcellulose 24 hours post-nucleofection. Leukemic CFU-L colonies were allowed to develop in clonogenic assays in methylcellulose and scored on day 6. Data are expressed as percentage of control untreated colonies and represent means ± S.E. of 5 independent experiments. <b>C</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid. Levels of SESN2 were quantified at 24 hours post-nucleofection by immunoblotting. <b>D</b>. KT-1 cells transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid were incubated in clonogenic assays in methylcellulose. Leukemic CFU-L colonies were scored on day 6 and data are expressed as percentage of control untreated colonies and represent means ± S.E. of 4 independent experiments. <b>E</b>. BV173R cells were transiently nucleofected with either empty vector (E.V.) or SESN2 or SESN3 expressing plasmid. 48 hours post-transfection, equal number of cells were plated and allowed to proliferate for 120 hours. Proliferation was measured by WST-1 assay at the indicated times. Data are expressed as the absorbance at 450 nm and represent means ± S.E. from 3 independent experiments, * p = 0.0018 comparing SESN3 nucleofected cells vs. E.V. nucleofected cells on day 4, ** p = 0.0068 comparing SESN3 nucleofected cells vs. E.V. nucleofected on day 5.</p

mTOR inhibition but not BCR-ABL inhibition upregulates SESN3 in T315I-BCR-ABL expressing cells.

<p><b>A</b>. BV173 or BV173R cells were treated with imatinib mesylate (5 µM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>B</b>. BV173 or BV173R cells were treated with either imatinib (5 µM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>C</b>. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with either imatinib (1 µM) or nilotinib (100 nM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>D</b>. BV173 and BV173R cells were treated with OSI-027 (5 µM) for 12 hours. RNA was extracted and expression of SESN3 mRNA was determined by quantitative RT-PCR, using GAPDH for normalization. Data are expressed as fold increase in the treated samples over untreated samples and represent means ± S.E. of 3 independent experiments. <b>E</b>. BV173 or BV173R cells were treated with OSI-027 (5 µM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>F</b>. Ba/F3 cells stably transfected with WT-BCR-ABL or T315I-BCR-ABL were treated with OSI-027 (5 µM) for 16 hours. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated.</p

Differential effects of SESN3 and SESN2 overexpression on mTOR and MAPK signaling effectors.

<p><b>A</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid and were analyzed for the presence of ROS by flow cytometry, following 30 minutes of staining with DCFDA at the time-points indicated. Data are as percent control empty vector for each time-point and represent means ± SE of 3 independent experiments. <b>B</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.) or SESN2 expressing plasmid. Cells were lysed 48 hours post-nucleofection Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN2 or GAPDH as indicated. <b>C</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid. Cells were lysed 24 hours post-nucleofection Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against SESN3 or GAPDH as indicated. <b>D</b>. KT-1 cells were transiently nucleofected with either empty vector (E.V.) or SESN3 expressing plasmid. Expression of mTOR was quantified at 24 hours post-nucleofection either by quantitative RT-PCR. Data are expressed as fold increase in the Sesn3-nucleofected samples over E.V.-nucleofected samples normalized to GAPDH and represent means ± S.E. of 3 independent experiments. <b>E</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.), SESN2 or SESN3 expressing plasmids. Cells were lysed 48 hours post-nucleofection and equal amounts of protein from cell lysates from the same experiment for each panel were resolved separately by SDS-PAGE and immunoblotted with the indicated antibodies. <b>F–G</b>. KT1 cells were transiently nucleofected with either empty vector (E.V.), SESN2 or SESN3 expressing plasmids. Cells were lysed 48 hours post-nucleofection and equal amounts of protein were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Blots were subsequently stripped and reprobed with the respective antibodies against the total form of the protein. Densitometry analysis of the representative blots is shown.</p

Reduction in VEGFR2, FGFR1, or FOXO1 expression in ADSCs/ECFCs reduced growth factor-driven <i>in vitro</i> cord formation.

<p>(AB); ADSCs/ECFCs were transduced with non-targeting (control), or pooled shRNA directed against VEGFR2, FOXO1, or FGFR1, for 72 hours and (A) whole cell protein extracts were isolated and subjected to Western blot analysis using antiserum against VEGFR2, FOXO1 and FGFR1 (82.1±5.9, 91.5±2.7 and 73.7±7.1% knockdown respectively), using β-actin as a loading control, or (B) analyzed for cord formation with PBS (Basal), 10 ng/ml VEGF, or 15 ng/ml bFGF stimulation for 72 hours before immunohistochemistry for CD31 (green), α-smooth muscle actin (red), and Hoechst to stain nuclei (blue). Representative images (5× magnification) are shown. Graphs represent mean ± standard error from three independent experiments, and asterisks denote statistically significant differences (*, <i>p</i><0.05; **, p<0.01; ***, p<0.001) compared to non-targeting shRNA controls.</p