Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma.

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

Effective suicide gene therapy for leukemia in a model of insertional oncogenesis in mice.

The safety of gene therapy using hematopoietic stem cells may be increased by including a suicide gene in the therapeutic vector to eliminate adverse events like insertional oncogenesis while retaining the clinical benefits. We have developed a model of experimental insertional oncogenesis by transducing the murine factor-dependent leukemia cell line Ba/F3 with a bicistronic Moloney murine leukemia virus retroviral vector encoding a murine oncogene (cKit(D814V)) in addition to one of three suicide genes: Herpes simplex virus thymidine kinase (HSV-TK); SR39, an HSV-TK mutant with an increased affinity for the drug substrate Ganciclovir (GCV); or sc39, a splice-corrected version of SR39. Following intravenous challenge with transduced Ba/F3 clones and treatment with GCV, leukemia developed in mice given cells expressing HSV-TK, but not SR39 or sc39. In vitro GCV resistance was observed in heterogeneously transduced Ba/F3 pools at 2.5-14%, and single-nucleotide changes or partial loss of the suicide gene were identified as mechanisms of drug escape. However, GCV treatment resulted in 80-100% survival of mice challenged even with pools of partially resistant Ba/F3 cells expressing SR39 or sc39. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity.

Loss of cellular adhesion to matrix induces p53-independent expression of PTEN tumor suppressor.

BACKGROUND: The tumor suppressor gene PTEN has been found mutated in many types of advanced tumors. When introduced into tumor cells that lack the wild-type allele of the gene, exogenous PTEN was able to suppress their ability to grow anchorage-independently, and thus reverted one of the typical characteristics of tumor cells. As these findings indicated that PTEN might be involved in the regulation of anchorage-dependent cell growth, we analyzed this aspect of PTEN function in non-tumor cells with an anchorage-dependent phenotype. RESULTS: We found that in response to the disruption of cell-matrix interactions, expression of endogenous PTEN was transcriptionally activated, and elevated levels of PTEN protein and activity were present in the cells. These events correlated with decreased phosphorylation of focal adhesion kinase, and occurred even in the absence of p53, a tumor suppressor protein and recently established stimulator of PTEN transcription. CONCLUSIONS: In view of PTEN's potent growth-inhibitory capacity, we conclude that its induction after cell-matrix disruptions contributes to the maintenance of the anchorage-dependent phenotype of normal cells.

Differential effects of selective COX-2 inhibitors on cell cycle regulation and proliferation of glioblastoma cell lines.

It is well established that traditional NSAIDs, which inhibit cyclooxygenase (COX) 1 and COX-2, have the potential to reduce the risk of colorectal cancer. New generation COX inhibitors have been developed that selectively inhibit COX-2, which might cause less side effects while still retaining their therapeutic potential. As patients with brain tumors, such as glioblastoma, exhibit a very poor prognosis, we began to explore whether COX inhibitors could be useful for the treatment of this type of tumor. We found that celecoxib inhibited the proliferation of various glioblastoma cell lines in vitro much more potently than traditional NSAIDs. In addition, although several different selective COX-2 inhibitors potently reduced PGE2 levels in these cells, none of them exerted anti-proliferative effects that were comparable to celecoxib. The addition of external PGE2 to celecoxib-treated cells did not restore proliferation, indicating that growth inhibition by celecoxib was not mediated via the blockage of PGE2 production. In an effort to determine the underlying molecular processes that might mediate celecoxib's potent anti-proliferative effects, we found a loss of the activity of cyclin-dependent kinases, the essential regulators of cell proliferation, which was due to the transcriptional downregulation of cyclin A and cyclin B expression. Taken together, our results show that celecoxib exerts COX-2-independent anti-proliferative effects on glioblastoma cell growth, which are more potent than those of other selective COX-2 inhibitors or traditional NSAIDs, and which are mediated via the transcriptional inhibition of two essential components of the cell cycle machinery, cyclin A and cyclin B.

Effect of reproductive hormones on ovarian epithelial tumors: I. Effect on cell cycle activity.

We examined the effects of the 4 major female reproductive hormones, estradiol (E2), progesterone (P4), follicle stimulating hormone (FSH), and luteinizing hormone (LH) on thymidine incorporation in benign and malignant ovarian epithelial tumors cultured in vitro. Treatment of these tumors with E2, FSH and LH resulted in increased thymidine incorporation while treatment with P4 inhibited growth as well as thymidine incorporation. The inhibitory effect of progesterone could not be reproduced by treating the cells with ligands for other steroid hormone receptors known to interact with P4 such as the mineralocorticoid and glucocorticoid receptors. All cells lines expressed at least the PR-A isoform of the progesterone receptor. ORG2058, R5020, RU486, and ZK98299 acted as progesterone receptor agonists with regard to their effect on thymidine incorporation. P4 down-regulated cyclin Bl expression and cdkl activity and up-regulated the p21 and p27 proteins. Expression of a reporter gene downstream to an AP-1 responsive element in a plasmid construct transfected into ovarian epithelial tumor cells was induced by P4 and inhibited by RU486. We conclude that P4 inhibits cell cycle activity in ovarian epithelial tumors, in part via down-regulation of the cdkl/cyclin B complex. This inhibitory effect may have therapeutic utility against ovarian epithelial tumors.

Suppression of the transformed phenotype and induction of differentiation-like characteristics in cultured ovarian tumor cells by chronic treatment with progesterone.

Epidemiological evidence suggests that elevated levels of the pregnancy hormone progesterone might play a role in the reduced risk of women to develop ovarian cancer. In vitro studies have supported this hypothesis by demonstrating negative effects of this hormone on the growth and proliferation of cultured ovarian carcinoma cells. However, little is known about the underlying molecular processes and how progesterone might decrease the risk for ovarian tumors. Therefore, we investigated the effects of chronic hormone treatment on the cell-cycle and transformed phenotype of ovarian carcinoma cell lines in vitro. We found that long-term treatment of these cells with progesterone caused a concomitant reduction of cyclin-dependent kinase (CDK) activity. In parallel, these cells lost their transformed phenotype as indicated by the acquisition of contact inhibition and the loss of anchorage-independence, as well as the reduced expression of tumor markers such as heat shock protein (HSP) 72 and carcinoma antigen (CA) 125. In addition, progesterone-treated cells exhibited characteristics that resembled a more differentiated phenotype. Taken together, our data indicated that progesterone was able to suppress the transformed phenotype of ovarian tumor cells. This observation could serve to explain progesterone's alleged protective effect in ovarian carcinogenesis.