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A genetically defined mouse ovarian carcinoma model for the molecular characterization of pathway-targeted therapy and tumor resistance.

ABSTRACT: Cell lines and tumors with defined genetic alterations provide ideal systems in which to test the molecular mechanisms of tumor sensitivity to pathway-targeted therapy. We have generated mouse ovarian epithelial tumor cell lines that contain various combinations of genetic alterations in the p53, c-myc, K-ras and Akt genes. Using both in vitro and in vivo approaches, we investigated the effect of rapamycin on cell proliferation, tumor growth, and the accumulation of peritoneal ascites. We demonstrated that rapamycin effectively inhibits the growth of tumors that rely on Akt signaling for proliferation, whereas tumors in which Akt signaling is not the driving force in proliferation are resistant to rapamycin. The introduction of activated Akt to the rapamycin-resistant cells does not render the cells susceptible to rapamycin if they can use alternative pathways for survival and proliferation. Accordingly, the rapamycin-sensitive tumors develop resistance to rapamycin when presented with alternative survival pathways, such as the mitogen-activated extracellular kinase signaling pathway. The combination of rapamycin and the mitogen-activated extracellular kinase inhibitor PD98059 is required to diminish proliferation in these cell lines. Our results indicate that mammalian target of rapamycin inhibitors may be effective in a subset of tumors that depend on Akt activity for survival but not effective in all tumors that exhibit Akt activation. Tumors with alternative survival pathways may require the inactivation of multiple individual pathways for successful treatment.

SUBMITTER: Xing D

PROVIDER: S-EPMC1087513 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ovarian carcinoma (OC) is the fifth leading cause of death among women in the United States. Persistent activation of signal transducer and activator of transcription (STAT3) is frequently detected in OC. STAT3 is activated by Janus family kinases (JAK) via cytokine receptors, growth factor receptor and non-growth factor receptor tyrosine kinases. Activation of STAT3 mediates tumor cell proliferation, survival, motility, invasion, and angiogenesis, and recent work demonstrates that STAT3 activation suppresses anti-tumor immune responses and supports tumor-promoting inflammation. We hypothesized that therapeutic targeting of the JAK/STAT3 pathway would inhibit tumor growth by direct effects on OC cells and by inhibition of cells in the tumor microenvironment (TME). To test this, we evaluated the effects of a small molecule JAK inhibitor, AZD1480, on cell viability, apoptosis, proliferation, migration and adhesion of OC cells in vitro. We then evaluated the effects of AZD1480 on in vivo tumor growth and progression, gene expression, tumor-associated matrix metalloproteinase (MMP) activity and immune cell populations in a transgenic mouse model of OC. AZD1480-treatment inhibited STAT3 phosphorylation and DNA binding, and migration and adhesion of cultured OC cells and ovarian tumor growth rate, volume and ascites production in mice. In addition, drug treatment led to altered gene expression, decreased tumor-associated MMP activity, and fewer suppressor T cells in the peritoneal tumor microenvironment of tumor-bearing mice than control mice. Taken together, our results show pharmacological inhibition of the JAK2/STAT3 pathway leads to disruption of functions essential for ovarian tumor growth and progression and represents a promising therapeutic strategy. 8 female C57BL/6JTgMISIIR-TAg mice with ovarian tumors of ~500 mm3 were used. 4 vehicle-treated mice (0.5% hypermellose/0.1%Tween 80);4 drug-treated mice (30 mg/kg AZD1480 in 0.5% hypermellose/0.1%Tween 80).

Project description:BACKGROUND:The phosphoinositol 3-kinase (PI3K) pathway is associated with poor prognosis of hematologic malignancies, providing a strong rationale for the use of PI3K inhibitors in the treatment of malignant lymphoma. However, development of resistance limits the use of PI3K inhibitors in lymphoma patients. METHODS:We established copanlisib (pan-PI3K inhibitor)-resistant B-cell lymphoma and duvelisib (PI3Kδ and -γ inhibitor)-resistant T-cell lymphoma cell lines. The cytokine array and the phospho-kinase array were used to identify up-regulated proteins in the resistant cells. Cytokine expression and phospho-kinase levels were examined by ELISA and Western blot analysis, respectively. Cell proliferation capabilities were measured by using CCK-8 kit and colony formation assay. The effects of inhibitors on apoptosis were detected using an Annexin V-FITC Apoptosis Detection Kit and a flow cytometry system. The underlying mechanisms were studied by transfecting recombinant plasmids or siRNA into lymphoma cell lines. Cells were transiently transfected using the Amaxa electroporation system. We evaluated the effects of PI3K inhibitor alone and in combination with JAK inhibitor (BSK805) on lymphoma proliferation and signaling pathway activation. RESULTS:Cytokine arrays revealed upregulation of interleukin (IL)-6 in both copanlisib- and duvelisib-resistant cell lines. Phosphorylated STAT5, AKT, p70S6K and MAPK were increased in copanlisib-resistant B-cell lymphoma cells, whereas phosphorylated STAT3 and NF-κB were increased in duvelisib-resistant T cell lymphoma cells. Conversely, depletion of IL-6 sensitized both resistant cell lines, and led to downregulation of phosphorylated STAT3 and STAT5 in copanlisib- and duvelisib-resistant cells, respectively. Moreover, combined treatment with a JAK inhibitor (BSK805) and a PI3K inhibitor circumvented the acquired resistance to PI3K inhibitors in lymphoma, and concurrent inhibition of the activated pathways produced combined effects. CONCLUSIONS:IL-6-induced STAT3 or STAT5 activation is a critical mechanism underlying PI3K inhibitor resistance in lymphoma, supporting the utility of IL-6 as an effective biomarker to predict therapeutic response to PI3K inhibitors.

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A genetically defined mouse ovarian carcinoma model for the molecular characterization of pathway-targeted therapy and tumor resistance.

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