MicroRNA-221/222 Confers Tamoxifen Resistance in Breast Cancer by Targeting p27\u3csup\u3eKip1\u3c/sup\u3e

We explored the role of microRNAs (miRNAs) in acquiring resistance to tamoxifen, a drug successfully used to treat women with estrogen receptor-positive breast cancer. miRNA microarray analysis of MCF-7 cell lines that are either sensitive (parental) or resistant (4-hydroxytamoxifen-resistant (OHTR)) to tamoxifen showed significant (50%) of seven miRNAs in OHT R cells compared with parental MCF-7 cells. Increased expression of three of the most promising up-regulated (miR-221, miR-222, and miR-181) and down-regulated (miR-21, miR-342, and miR-489) miRNAs was validated by real-time reverse transcription-PCR. The expression of miR-221 and miR-222 was also significantly (2-fold) elevated in HER2/neu-positive primary human breast cancer tissues that are known to be resistant to endocrine therapy compared with HER2/neu-negative tissue samples. Ectopic expression of miR-221/222 rendered the parental MCF-7 cells resistant to tamoxifen. The protein level of the cell cycle inhibitor p27Kip1, a known target of miR-221/222, was reduced by 50% in OHTR cells and by 28-50% in miR-221/222-overexpressing MCF-7 cells. Furthermore, overexpression of p27Kip1 in the resistant OHT R cells caused enhanced cell death when exposed to tamoxifen. This is the first study demonstrating a relationship between miR-221/222 expression and HER2/neu overexpression in primary breast tumors that are generally resistant to tamoxifen therapy. This finding also provides the rationale for the application of altered expression of specific miRNAs as a predictive tamoxifen-resistant breast cancer marker

Methylation of the PTPRO gene in human hepatocellular carcinoma and identification of VCP as its substrate

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type-O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate-trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate-trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild-type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy

Human Genetic Relevance and Potent Antitumor Activity of Heat Shock Protein 90 Inhibition in Canine Lung Adenocarcinoma Cell Lines

<div><p>Background</p><p>It has been an open question how similar human and canine lung cancers are. This has major implications in availability of human treatments for dogs and in establishing translational models to test new therapies in pet dogs. The prognosis for canine advanced lung cancer is poor and new treatments are needed. Heat shock protein 90 (HSP90) is an ATPase-dependent molecular chaperone ubiquitously expressed in eukaryotic cells. HSP90 is essential for posttranslational conformational maturation and stability of client proteins including protein kinases and transcription factors, many of which are important for the proliferation and survival of cancer cells. We investigated the activity of STA-1474, a HSP90 inhibitor, in two canine lung cancer cell lines, BACA and CLAC.</p><p>Results</p><p>Comparative genomic hybridization analysis of both cell lines revealed genetic relevance to human non-small cell lung cancer. STA-1474 inhibited growth and induced apoptosis of both cell lines in a dose- and time-dependent manner. The ICs₅₀ after 72 h treatment with STA-1474 were 0.08 and 0.11 μM for BACA and CLAC, respectively. When grown as spheroids, the IC₅₀ of STA-1474 for BACA cells was approximately two-fold higher than when grown as a monolayer (0.348 μM <i>vs</i>. 0.168 μM), whereas CLAC spheroids were relatively drug resistant. Treatment of tumor-stromal fibroblasts with STA-1474 resulted in a dose-dependent decrease in their relative cell viability with a low IC₅₀ of 0.28 μM.</p><p>Conclusions</p><p>Here we first established that lung adenocarcinoma in people and dogs are genetically and biochemically similar. STA1474 demonstrated biological activity in both canine lung cancer cell lines and tumor-stromal fibroblasts. As significant decreases in relative cell viability can be achieved with nanomolar concentrations of STA-1474, investigation into the clinical efficacy of this drug in canine lung cancer patients is warranted.</p></div

Viability assays and half-maximal inhibitory concentrations (ICs₅₀) for canine lung cancer cell lines.

<p>Cells were treated with increasing concentrations cytotoxic drugs (A) or small molecules inhibitors (B) and proliferation was evaluated after 72 h. Treatment effects were normalized to the drug vehicle-treated control group. Each graph shows mean±SEM. ICs₅₀ were calculated for each experiment. The dotted line in the y-axis represents the 50% relative viability. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Characterization of BACA and CLAC canine lung cancer lines by RT-PCR.

<p>(A) Reverse transcriptase cDNA transcripts of NKX2-1 (lung cancer cell marker) and HSP90 client proteins in BACA and CLAC cell lines. NDUFA1 serves as a loading control. (B) Reverse transcriptase cDNA transcripts of HSP70 and three HSP90 isoforms in BACA and CLAC cell lines. NDUFA1 serves as a loading control.</p

Gene annotation summary of focal copy number alterations in BACA and CLAC.

<p>Gene annotation summary of focal copy number alterations in BACA and CLAC.</p

Protein expression of HSP90-regulated proteins, HSP70 and HSP90 in canine lung cancer cell lines after treatment with small molecule inhibitors.

<p>A set of three plates for each cell line was used for evaluation of total and phosphoproteins of downstream signaling pathways (A) Representative immunoblots of HSP90 client protein expression from BACA whole cell protein lysates after treatment with STA-1474 or toceranib phosphate. (B) Representative immunoblots of HSP90 client protein expression from CLAC whole cell protein lysates after treatment with STA-1474 or toceranib phosphate. Controls were cell lines treated with the drug solvent, DMSO, as represented by the 0 concentration. Evaluation of phosphoprotein forms of the proteins are indicated by “p”. Drug concentrations are μmol/L. The β-actin Western blots serve as loading controls. (C) Immunobloting from whole cell protein lysates of HSP70 and HSP90 of BACA and CLAC lines treated with DMSO (control), STA-1474 and toceranib phosphate.</p

Viability and half-maximal inhibitory concentration (ICs₅₀) of STA-1474 treated canine lung cancer cell-line derived tumor spheroids, monolayers and tumor-stromal fibroblasts.

<p>(A) Tumor spheroids derived from both cell lines were allowed to form for 72 h after seeding cells in the ultra-low attachment wells. Images taken of one field of view/well on an inverted microscope at 40X magnification, scale bar represents 200 μM (B) Immediately after formation, tumor spheroids were treated with DMSO or increasing concentrations of STA-1474 for 72 h. Tumor spheroids and monolayers from each cell line were grown and treated identically. Cultured cells were treated with STA-1474 for an additional 72 h and ICs₅₀ were calculated. (C) Tumor-stromal fibroblasts were seeded, allowed to form a monolayer for 24 h then incubated with STA-1474 for 72 h and viability determined. Experiments were performed in four replicates and repeated twice. Each graph shows mean ± SEM and each group was compared to DMSO. The dotted line in the y-axis represents the 50% relative viability. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Relative cell viability assays, half-maximal inhibitory concentrations (ICs₅₀) after treatment with an HSP70 inhibitor (VER155008) or a combination of STA-1474 and VER155008.

<p>(A) Cells were treated with HSP70 inhibitor, (VER155008) for 72 h. The relative viability and ICs₅₀ were determined for both cell lines after treatment. (B) BACA or CLAC cells were plated in DMEM media for 24 h and then treated for 72 h with VER155008 only (gray squares), STA-1474 only (black circles) or a combination of VER155008 and STA-1474 concentrations (red triangles) ranging from 0.0625X to 16X their IC₅₀ concentrations. Each graph shows mean ± SEM. Each group was compared to the DMSO control. The dotted line in the y-axis represents the 50% relative viability. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001. (C) Multi-drug combination dose-effect analysis for the doublet combination of VER155008 and STA-1474 on BACA and CLAC cell lines as measured by the combination index (CI). The CI value definitions are represented in the description column.</p