An inhibitor of HIV-1 protease modulates constitutive eIF2α dephosphorylation to trigger a specific integrated stress response.

Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity.

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1ß. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.

Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.

A2780 human ovarian carcinoma cells respond to treatment with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) with the production of dihydroceramide and with a concomitant reduction of cell proliferation and induction of apoptosis. The derived HPR-resistant clonal cell line, A2780/HPR, is less responsive to HPR in terms of dihydroceramide generation. In this report, we show that the production of sphingosine 1-phosphate (S1P) is significantly higher in A2780/HPR versus A2780 cells due to an increased sphingosine kinase (SK) activity and SK-1 mRNA and protein levels. Treatment of A2780 and A2780/HPR cells with a potent and highly selective pharmacological SK inhibitor effectively reduced S1P production and resulted in a marked reduction of cell proliferation. Moreover, A2780/HPR cells treated with a SK inhibitor were sensitized to the cytotoxic effect of HPR, due to an increased dihydroceramide production. On the other hand, the ectopic expression of SK-1 in A2780 cells was sufficient to induce HPR resistance in these cells. Challenge of A2780 and A2780/HPR cells with agonists and antagonists of S1P receptors had no effects on their sensitivity to the drug, suggesting that the role of SK in HPR resistance in these cells is not mediated by the S1P receptors. These data clearly demonstrate a role for SK in determining resistance to HPR in ovarian carcinoma cells, due to its effect in the regulation of intracellular ceramide/S1P ratio, which is critical in the control of cell death and proliferation.

Overexpression of epidermal growth factor type-1 receptor (EGF-R1) in cervical cancer: implications for Cetuximab-mediated therapy in recurrent/metastatic disease.

OBJECTIVES: To evaluate and compare epidermal growth factor type-1 receptor (EGF-R1) expression in short term and established cervical cancer cell lines generated from primary and metastatic/recurrent sites of disease. To evaluate the sensitivity of cervical cancer cell lines to treatment with a chimeric MAb against EGFR-1 (Cetuximab). METHODS: EGFR-1 expression was evaluated by flow cytometry on 22 cervical cancer cell lines including 14 primary cervical cancer cell lines obtained from cervical biopsies (11 patients) and recurrent sites of disease (three patients) as well as eight established cell lines. Tumor cell lines were tested for sensitivity to Cetuximab-mediated complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in 51Cr release assays. Finally, Cetuximab-mediated inhibition of cell proliferation was also tested. RESULTS: Fourteen out of fourteen (100%) primary tumors and seven out of eight (87.5%) established cervical cancer cell lines expressed EGFR-1 by flow cytometry. Cell lines from recurrent/metastatic sites of disease expressed higher levels of EGFR-1 when compared to those obtained from primary sites (p>0.05). Minimal CDC was detected in the majority of cervical cancer cell lines exposed to complement+/-Cetuximab in the absence of peripheral blood lymphocytes (PBL). In contrast, cervical tumor cell lines were found highly sensitive to Cetuximab-mediated ADCC when challenged with PBL from either healthy donors or cervical cancer patients. Importantly, ADCC was further increased in the presence of complement. Finally, tumor proliferation was significantly inhibited by Cetuximab in all cervical tumors tested. CONCLUSIONS: EGFR-1 is highly expressed in primary and recurrent cervical tumors. Cetuximab might be a novel and attractive therapeutic strategy in patients harboring chemotherapy-resistant, recurrent, or metastatic cervical cancer.