Mice Null for the Deubiquitinase USP18 Spontaneously Develop Leiomyosarcomas

USP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease

Additional file 2: of Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas

Mice Null for the Deubiquitinase USP18 Spontaneously Develop Leiomyosarcoma. (DOCX 115 kb

Additional file 1: Table S1. of Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas

Antibodies used for immunohistochemistry studies. Table S2. Immunohistochemical staining of USP18 in clinical leiomyosarcoma samples. Figure S1. A: Dystrophic calcifications in USP18 null mice. A representative image of a USP18-/- mouse is shown. B: KHC-2 cells with reconstituted USP18 expression maintained expression for the duration of growth in mice. Immunoblot analysis of protein isolated from 4 control and 4 USP18 overexpressing independent orthotopic sarcomas harvested from mice. The immunoblot showed representative analysis of KHC-2 cells and this finding was also seen in KHC-1 cells (data not shown). Figure S2. USP18 null leiomyosarcoma cell lines are sensitive to treatment with the JAK2-STAT3 inhibitor, JSI-124. A: Immunoblot analysis of pSTAT3, STAT3, CDK4 and USP18 levels in KHC-1 cells with and without stably restored USP18 activity. B: Growth analysis of KHC-1 cells with JAK2-STAT3 inhibitor, JSI-124. Similar effects were seen in KHC-2 cells (data not shown). Validation of JSI-124 repression of JAK2-STAT3 pathway C: Immunoblot analyses of phosphorylated JAK2 (pJAK2), JAK2, and actin with relative level of pJAK2/JAK2 calculated relative to control. D: Immunoblot analysis of phosphorylated STAT3 (pSTAT3), STAT3, cyclin D1 and actin levels. Figure S3. USP18 null leiomyosarcoma cell line KHC-1 and human leiomyosarcoma cell line SK-LMS-1 growth in response to interferon-β (500Units/ml IFNB) or doxycycline (0.2ΟM Dox) treatment over 3 days. Results expressed as fold relative to vehicle treated cells. Each experiment was performed in triplicate 3 separate times. Figure S4. USP18 null leiomyosarcoma cell line KHC-1 with restored USP18 expression did not affect response to IFNb (500Units/ml). Results expressed as fold relative to vehicle treated cells. (N.S. = not significant). (PPTX 2131 kb

Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations

Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation