High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.

Glycogen synthase kinase-3ß inhibition ameliorates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice.

Decreased heart rate variability (HRV) is a major risk factor for sudden death and cardiovascular disease. We previously demonstrated that parasympathetic dysfunction in the heart of the Akita type 1 diabetic mouse was due to a decrease in the level of the sterol response element-binding protein (SREBP-1). Here we demonstrate that hyperactivity of glycogen synthase kinase-3ß (GSK3ß) in the atrium of the Akita mouse results in decreased SREBP-1, attenuation of parasympathetic modulation of heart rate, measured as a decrease in the high-frequency (HF) fraction of HRV in the presence of propranolol, and a decrease in expression of the G-protein coupled inward rectifying K(+) (GIRK4) subunit of the acetylcholine (ACh)-activated inward-rectifying K(+) channel (IKACh), the ion channel that mediates the heart rate response to parasympathetic stimulation. Treatment of atrial myocytes with the GSK3ß inhibitor Kenpaullone increased levels of SREBP-1 and expression of GIRK4 and IKACh, whereas a dominant-active GSK3ß mutant decreased SREBP-1 and GIRK4 expression. In Akita mice treated with GSK3ß inhibitors Li(+) and/or CHIR-99021, Li(+) increased IKACh, and Li(+) and CHIR-99021 both partially reversed the decrease in HF fraction while increasing GIRK4 and SREBP-1 expression. These data support the conclusion that increased GSK3ß activity in the type 1 diabetic heart plays a critical role in parasympathetic dysfunction through an effect on SREBP-1, supporting GSK3ß as a new therapeutic target for diabetic autonomic neuropathy.

Adrenergic differentiation and Ret expression in rat pheochromocytomas.

Pheochromocytomas are catecholamine-producing tumors of the adult adrenal medulla. They are rare in humans and most other species but common in laboratory rats. However, the relevance of rat pheochromocytomas as a model for their human counterparts is uncertain. Previous studies of spontaneous and drug-induced rat pheochromocytomas and the PC12 pheochromocytoma cell line suggested a distinctive noradrenergic phenotype, possibly reflecting origin from a progenitor not present in the adult human adrenal. In this study, we studied 31 pheochromocytomas derived from test and control male and female rats in toxicologic studies for expression of the epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) and the receptor tyrosine kinase Ret. PNMT, which defines adrenergic chromaffin cells, is frequently expressed in human pheochromocytomas, often in tumors that also overexpress RET. We also tested for the expression of the cell cycle checkpoint protein p27(Kip1), which recently was reported absent in pheochromocytomas from a strain of rats with a hereditary mixed multiple endocrine neoplasia (MEN)-like syndrome. Using immunoblots, we demonstrated PNMT expression in almost 50% of the 31 tumors, although often at lower levels than in normal rat adrenal medulla. The majority of tumors overexpressed Ret. There was no apparent correlation between PNMT and Ret. However, in this study, PNMT expression was strongly associated with tumors arising in female rats, while overexpression of Ret did not show a sex predilection. Robust expression of p27(Kip1) was seen in all tumors from the toxicologic studies and also in a small sample of pheochromocytomas from Long-Evans rats, which were reported to have a mixed MEN-like syndrome in the 1980s. The present results show that rat pheochromocytomas have greater phenotypic diversity than previously believed and greater similarity to their human counterparts with respect to these two important markers. Loss of p27(Kip1) does not appear to account for the high frequency of pheochromocytomas in commonly utilized rat strains.

ErbB-2 induces bilateral adrenal pheochromocytoma formation in mice.

Pheochromocytoma (PCC) is a rare catecholamine-producing tumor that arises from the adrenal medulla and is often familial. The genetic basis for familial PCC involves mutations of RET, VHL, SHDx or NF-1 in more than 20% of cases. Additional genes may be important in pathogenesis of both familial and sporadic PCC. ErbB-2/Her2/Neu is a growth factor receptor tyrosine kinase that is frequently overexpressed in tumors and there is clinical evidence suggesting that enhanced ErbB-2 growth factor receptor signaling may play a role in PCC. In the present study, ectopic expression of an activated ErbB-2 transgene resulted in bilateral adrenal PCC. Analyses of tumor samples and normal adrenal tissue revealed that levels of the Pten tumor suppressor protein were greatly reduced in PCCs, while levels of the cell cycle regulatory protein cyclin D1 were usually increased. In addition, levels of phospo-AKT were increased in PCCs versus normal adrenal tissue. Biochemical analyses established that PCC's were functionally active, producing abundant levels of the catecholamines, epinephrine and norepinephrine. These data establish that increased ErbB-2 growth factor receptor signaling in the adrenal medulla can lead to PCC through combined influences on Pten, AKT andcyclin D1.