Targeting Lactate Dehydrogenase-A Inhibits Tumorigenesis and Tumor Progression in Mouse Models of Lung Cancer and Impacts Tumor-Initiating Cells

The lactate dehydrogenase-A (LDH-A) enzyme catalyzes the interconversion of pyruvate and lactate, is upregulated in human cancers, and is associated with aggressive tumor outcomes. Here we use an inducible murine model and demonstrate that inactivation of LDH-A in mouse models of NSCLC driven by oncogenic K-RAS or EGFR leads to decreased tumorigenesis and disease regression in established tumors. We also show that abrogation of LDH-A results in reprogramming of pyruvate metabolism, with decreased lactic fermentation in vitro, in vivo, and ex vivo. This was accompanied by reactivation of mitochondrial function in vitro, but not in vivo or ex vivo. Finally, using a specific small molecule LDH-A inhibitor, we demonstrated that LDH-A is essential for cancer-initiating cell survival and proliferation. Thus, LDH-A can be a viable therapeutic target for NSCLC, including cancer stem cell-dependent drug-resistant tumors

New stimulators and a new mechanism of regulated secretion in pituitary gonadotropes

Thesis (Ph. D.)--University of Washington, 1997The major regulator of gonadotropin (luteinizing hormone, LH; and follicle-stimulating hormone, FSH) secretion is the peptide gonadotropin-releasing hormone (GnRH). It acts via a rise in intracellular Ca\sp{2+} concentration ( (Ca\sp{2+}\rbrack\sb{\rm i}). I used cultured pituitary gonadotropes of male rats to seek other possible agents and mechanisms that might regulate gonadotropin secretion. The GnRH-evoked (Ca\sp{2+}\rbrack\sb{\rm i} rise is oscillatory and accompanied by oscillations of outward K\sp{+} current that is activated by Ca\sp{2+}. Substances that caused current responses similar to those with GnRH were hypothesized to evoke gonadotropin secretion via the Ca\sp{2+}-dependent mechanism. Endothelin-1, oxytocin, neurotensin, pituitary adenylate cyclase-activating polypeptide, and serotonin raised (Ca\sp{2+}\rbrack\sb{\rm i} and evoked LH release as assayed by the reverse hemolytic plaque assay (RHPA). These agents affected only subpopulations of gonadotropes establishing functional heterogeneity of pituitary gonadotropes. On the other hand, a protein kinase C (PKC) activator--phorbol-12-myristate-13-acetate (PMA)--caused LH release from pituitary gonadotropes without evoking changes in (Ca\sp{2+}\rbrack\sb{i}. After 1 hour incubation in RHPA, PMA elicited an increase in LH secretion comparable to that caused by GnRH. A PKC inhibitor did not alter GnRH-induced LH release indicating that PKC activation is not important for GnRH-induced secretion in a 1 hour application. Buffering of intracellular Ca\sp{2+} by AM-loaded BAPTA reduced GnRH-induced LH secretion almost to control levels without significantly affecting PMA-induced secretion. Single-cell photometry on gonadotropes previously identified by incubation with GnRH in RHPA, revealed no changes in (Ca\sp{2+}\rbrack\sb{\rm i} during a one hour application of PMA. Hence, there appears to be a Ca\sp{2+}-independent pathway of regulated secretion in gonadotropes. Its nature and physiological ligands remain to be elucidated. Finally, I attempted to develop a technique to study differential regulation of LH and FSH release. This attempt failed due to methodological problems but the method may allow study of secretion of two hormones from cell systems other than pituitary gonadotropes. Thus, gonadotropes possess several mechanisms of regulated secretion and can be regulated by a variety of neuromodulators, suggesting possible ways of differential regulation of LH and FSH secretion

The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis

Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-beta1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE(2) increased apoptosis threefold, while treatment of pre-osteocytes with TGF-beta1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival