MicroRNA 17-92 cluster mediates ETS1 and ETS2-dependent RAS-oncogenic transformation.

The ETS-family transcription factors Ets1 and Ets2 are evolutionarily conserved effectors of the RAS/ERK signaling pathway, but their function in Ras cellular transformation and biology remains unclear. Taking advantage of Ets1 and Ets2 mouse models to generate Ets1/Ets2 double knockout mouse embryonic fibroblasts, we demonstrate that deletion of both Ets1 and Ets2 was necessary to inhibit HrasG12V induced transformation both in vitro and in vivo. HrasG12V expression in mouse embryonic fibroblasts increased ETS1 and ETS2 expression and binding to cis-regulatory elements on the c-Myc proximal promoter, and consequently induced a robust increase in MYC expression. The expression of the oncogenic microRNA 17-92 cluster was increased in HrasG12V transformed cells, but was significantly reduced when ETS1 and ETS2 were absent. MYC and ETS1 or ETS2 collaborated to increase expression of the oncogenic microRNA 17-92 cluster in HrasG12V transformed cells. Enforced expression of exogenous MYC or microRNA 17-92 rescued HrasG12V transformation in Ets1/Ets2-null cells, revealing a direct function for MYC and microRNA 17-92 in ETS1/ETS2-dependent HrasG12V transformation

Adenovirus and Oxaliplatin cooperate as agnostic sensitizers for immunogenic cell death in colorectal carcinoma

Treatments with cytotoxic agents or viruses may cause Immunogenic Cell Death (ICD) that immunize tumor-bearing hosts but do not cause complete regression of tumor. We postulate that combining two ICD inducers may cause durable regression in immunocompetent mice. ICD was optimized in vitro by maximizing calreticulin externalization in human colorectal carcinoma (CRC) cells by exposure to mixtures of Oxaliplatin (OX) and human adenovirus (AdV). Six mm diameter CT26 or 4T1 carcinomas in flanks of BALB/c mice were injected once intratumorally (IT) with OX, AdV or their mixture. Tumor growth, Tumor-Infiltrating Lymphocytes (TIL), nodal cytotoxicity, and rejection of a viable cell challenge were measured. Tumors injected IT once with an optimum mixture of 80 µM OX – AdV 25 Multiplicity of Infection (MOI) in PBS buffer were 17–29% the volume of control tumors. When buffer was changed from PBS to 5% dextrose in water (D5W), volumes of tumors injected IT with 80 µM OX-AdV 25 MOI were 10% while IT OX or AdV alone were 32% and 40% the volume of IT buffer-treated tumors. OX-AdV IT increased CD3+ TIL by 4-fold, decreased CD8+ PD-1+ TIL from 79% to 19% and induced cytotoxicity to CT26 cells in draining node lymphocytes while lymphocytes from CT26-bearing untreated mice were not cytotoxic. OX-AdV IT in D5W caused complete regression in 40% of mice. Long-term survivors rejected a contralateral challenge of CT26. The buffer for Oxaliplatin is critical. The two ICD inducer mixture is promising as an agnostic sensitizer for carcinomas like colorectal carcinoma

<i>Ets1</i>, <i>Ets2</i> and <i>c-Myc</i> activate miR17-92 transcription.

<p><i>C-Myc^f/f</i> MEFs were infected with <i>pBabe-Cre</i> retroviral vector or control <i>pBabe-empty</i> vector, selected with puromycin before functional examination. A) Protein analysis of 20 µg lysates from MEFs by western blot using antibodies as indicated B) Relative expression of <i>Ets1</i>, <i>Ets2</i> and <i>c-Myc</i> mRNAs after 48 hrs of transfection of expression vector. Asterisk represents P<0.05. C and D) Fold change relative real time PCR gene expression of pre-<i>mir17-92</i> (C) and individual miRs of the cluster (D) after 48 hrs transient transfection of the indicated vectors in <i>c-Myc^−/−</i> MEFs relative to control empty vector. Asterisk indicates P<0.05.</p

<i>Ets1</i> and <i>Ets2</i> activate <i>c-Myc</i> expression in <i>Hras^G12V</i> transformed MEFs.

<p>A) Fold change of <i>c-Myc</i> gene expression by real time rt-PCR in the 4 different genetic groups examined. B) Western blotting analysis of 20 µg protein lysates probed with antibodies against MYC and α-tubulin. C) Schematic illustration of the <i>c-Myc</i> promoter showing conserved Ets binding sites (box) and the ChIP primers used (arrows) relative to the P2 promoter. D) ChIP performed on MEFs with indicated genotypes using anti-ETS1 and ETS2 antibodies with IgG as control. The threshold value for the promoter being studied was normalized to that of input values and represented as relative enrichment. Asterisk indicates P<0.05.</p

Deletion of both <i>Ets1</i> and <i>Ets2</i> ablates <i>Hras^G12V</i> transformation of MEFs.

<p><i>E1+E2+</i> and <i>E1−E2−</i> MEFs were infected with <i>HRasv^G12V</i> or empty vector control retrovirus, then selected with Hygromycin for at least 5 days before functional examination of the four generated cellular genotypes, as shown. A) Western blotting analysis of 20 µg protein lysates probed with antibodies against proteins shown, with α-tubulin as loading control. B) Representative images and C) Quantification of the cellular colonies that grew in soft agar from the four different genetic groups Asterisk indicates P<0.05 as determined by the Student <i>t</i> test. D) Cells were injected subcutaneously into nude mice (10⁶ cells per injection site) and after 3 weeks the tumors were harvested. The ratios represent the percentage of tumors that grew from the total number of injections for each of the different genotypes.</p

Overexpression of <i>c-Myc</i> in <i>Ets1/Ets2</i>-null MEFs rescued <i>Hras^G12V</i> transformation.

<p><i>E1−E2−</i> control and <i>E1−E2−/H-Rasv^G12V</i> cells were infected with either MSCV-<i>GFP</i> empty control or MSCV-<i>GFP-c-Myc</i> vector, and cells were sorted for GFP expression by FACS. A) 20 µg of protein lysates from <i>E1−E2−/H-Rasv12/MSCV-GFP control</i> and <i>E1−E2−/H-Rasv12/MSCV-GFP-c-Myc</i> cells analyzed by western blot against MYC and β-Actin (protein loading control) antibodies. B) Graph representing the percentage of tumors formed over the total number of injections for the indicated cellular genotypes. N/A indicates that there were no tumors observed for the specified group. C) Representative pictures of all tumors derived from <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-GFP-c-Myc</i> cells. D) Graph showing individual and average volumes of <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-GFP-c-Myc</i> tumors. Asterisk indicates P<0.05.</p

<i>mir17-92</i> expression depends on <i>Ets1</i> and <i>Ets2</i> in <i>Hras^G12V</i> transformed MEFs.

<p>A) Fold change of pre-<i>mir17-92</i> and B) individual <i>microRNAs</i> of the <i>mir17-92</i> cluster: in the indicated genetic groups relative to control cells. Asterisk indicates P<0.05. C) Schematic illustration of the <i>mir17-92</i> promoter showing conserved ETS(box) and MYC (triangle) binding sites, as well as the ChIP primers used (arrows) relative to the start RNA start site. D) ChIP performed on the indicated MEFs genotypes using IgG control and specific antibodies as indicated. Asterisk indicates P<0.05.</p

<i>MiR-17-92</i> overexpression in <i>Ets1/Ets2</i>-null MEFs rescue <i>Hras^G12V</i> transformation.

<p><i>E1−E2−/pBabe</i> control and <i>E1−E2−/H-Rasv12</i> were infected with either MSCV-puro empty control or MSCV-puro-<i>miR-17- 92</i> vector and cells were selected by Puromycin before further functional analysis. A) pre-<i>mir17-92</i> cluster expression in the indicated genotypes relative to control vector. Asterisk indicated P<0.05. B) Graph demonstrating the percentage of tumors formed over the total number of injections for the different cellular groups. N/A indicates that there were no tumors observed for the specified group. (C) Representative images showing the total <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-puro-miR-17-92</i> derived tumors. D) Graph indicating individual and average volume of <i>E1+E2+</i>/<i>H-Rasv12</i> and <i>E1−E2−/H-Rasv12/MSCV-puro-miR-17-92</i> tumors.</p