Regulation of the production of the biologically active vitamin D3 sterol 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by cultured pulmonary alveolar macrophages (PAM) obtained from 6 patients with pulmonary sarcoidosis and from 9 normal subjects was studied. The sarcoid cells, all collected from patients with normal calcium metabolism, synthesized 1,25-(OH)2-[3H]D3 from the substrate 25-hydroxyvitamin [3H]D3 (25OH-[3H]D3), whereas in vitro incubation with recombinant human interferon-gamma (IFN gamma) or lipopolysaccharide (LPS) was required for induction of synthesis of the hormone by normal PAM. Exogenous 1,25-(OH)2D3 (10-100 nmol/L) decreased endogenous hormone production by normal PAM by approximately 45%. The relative inhibitory effect of 1,25-(OH)2D3 was less pronounced in sarcoid PAM, in which 10-100 nmol/L 1,25-(OH)2D3 inhibited 250HD3-1-hydroxylase by approximately 25%. An accompanying induction of the 250HD3-24-hydroxylase, which is typical for renal cells, was found at low levels in only 3 of 10 experiments; in this regard, no differences between sarcoid and normal PAM were apparent. PTH or forskolin did not influence 250HD3 metabolism by PAM. 1,25-(OH)2D3 production by sarcoid PAM was enhanced by lipopolysaccharide and IFN gamma. Likewise, recombinant human interleukin-2 stimulated 1,25-(OH)2D3 production by sarcoid PAM, suggesting a possible role for both IFN gamma and interleukin-2 in the induction of 1,25-(OH)2D3 synthesis by sarcoid PAM in vivo. Recombinant human IFN alpha, IFN beta, and granulocyte-macrophage colony-stimulating factor had little effect. Dexamethasone and chloroquine, which have in vivo antihypercalcemic activity in sarcoidosis, both inhibited 1,25-(OH)2D3 synthesis by sarcoid PAM; chloroquine simultaneously stimulated the 24-hydroxylase. Our studies suggest that the 250HD3-metabolizing system in PAM is in some respects different from renal metabolism of 250HD3.

The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the endoplasmic reticulum (ER), regulates the expression and activity of molecules including BiP (HSPA5), IRE1 (ERN1), Blimp-1 (PRDM1), and X-box binding protein 1 (XBP1). These molecules are required for terminal differentiation of B cells into plasma cells and expressed at high levels in plasma cell-derived multiple myeloma. Although these molecules have no known role at early stages of B-cell development, here we show that their expression transiently peaks at the pre-B-cell receptor checkpoint. Inducible, Cre-mediated deletion of Hspa5, Prdm1, and Xbp1 consistently induces cellular stress and cell death in normal pre-B cells and in pre-B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL1-and NRAS G12D oncogenes. Mechanistically, expression and activity of the UPR downstream effector XBP1 is regulated positively by STAT5 and negatively by the B-cellspecific transcriptional repressors BACH2 and BCL6. In two clinical trials for children and adults with ALL, high XBP1 mRNA levels at the time of diagnosis predicted poor outcome. A small molecule inhibitor of ERN1-mediated XBP1 activation induced selective cell death of patient-derived pre-B ALL cells in vitro and significantly prolonged survival of transplant recipient mice in vivo. Collectively, these studies reveal that pre-B ALL cells are uniquely vulnerable to ER stress and identify the UPR pathway and its downstream effector XBP1 as novel therapeutic targets to overcome drug resistance in pre-B ALL.

The PTEN protein is a negative regulator of the Akt pathway, leading to suppression of apoptosis and increased cell survival. Its role as a tumor-suppressor gene has been adequately substantiated, and homozygous mutations have been demonstrated in familial and sporadic cancers. In breast cancers, expression of PTEN protein is lost/reduced in 38% of cases. Somatic mutations are, however, rarely found. Our study was therefore designed to determine if differential methylation of the PTEN promoter region has a role in the transcriptional inactivation of the gene in invasive breast carcinomas. PTEN (MMAC1/TEP1) is a tumor-suppressor gene located on chromosomal subband 10q23.3. 1 It has been implicated in a number of familial and sporadic cancers. 2 Germline mutations have been reported in Cowden syndrome and Bannayan-Zonana syndrome, 2 dominantly inherited conditions causing predisposition to a variety of cancers including breast cancer. 3-5 Homozygous somatic mutations have also been recorded in a high percentage of human tumors, common among which are glioblastoma multiforme 6 -8 and endometrial cancer. 9 -11 Genetic, functional and animal modeling studies have substantiated the tumor-suppressor function of PTEN. The gene encodes a PIP3 phosphatase and regulates negatively the PIP3-Akt pathway, leading to suppression of apoptosis and increased cell survival. [12][13][14][15] PTEN is involved in breast cancers. We have previously demonstrated that expression of PTEN protein is lost/reduced in 38% of invasive breast cancers, a feature that is seen with highest frequency in high-grade carcinomas. 16 Although breast cancers also show 38% LOH at the PTEN locus, 17 somatic mutations have rarely been documented. [17][18][19] Thus, the events leading to loss of protein expression remain unclear. The discrepancy noted in the high incidence of LOH and/or protein loss and low mutation rates points to the possibility of epigenetic mechanisms in the inactivation of PTEN.The importance of epigenetic changes in human cancers is only now being recognized. Of primary interest is aberrant promoter hypermethylation that leads to inappropriate gene silencing. Hypermethylation of a growing number of genes (Rb, p16 INK4a , VHL, E-cadherin, APC and BRCA1, to name a few) occurs in promoter CpG islands in tumor cells and is being revealed as one of the most frequent mechanisms of loss of gene function. 20,21 Epigenetic changes are believed to occur at a higher frequency than genetic changes. The PTEN promoter region has been isolated, 22 and preliminary studies have identified methylation as a mechanism for PTEN inactivation in lung carcinomas. 23 Our study was therefore designed to determine if epigenetic regulation by differential methylation has a role in the transcriptional inactivation of the PTEN gene in invasive breast carcinomas. Normal breast tissue was also tested for methylation of the PTEN promoter and its relationship, if any, to aging. MATERIAL AND METHODSArchived tissue blocks from surgical specimens of 50 patients with in...

Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1α inhibitors. IRE1α-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1α-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1α inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1α inhibitor is a novel therapeutic approach for treatment of pancreatic cancers.