Vitamin D Resistance inRAS-Transformed Keratinocytes: Mechanism and Reversal Strategies

Abstract

Human retinoid X receptor alpha (hRXRalpha) plays a critical role in DNA binding and transcriptional activity through its heterodimeric association with several members of the nuclear receptor superfamily, including the vitamin D receptor (VDR). Several cancer cell lines derived from different tissues have been shown to be resistant to the growth-inhibitory action of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the biologically active metabolite of vitamin D(3). Here we show that in RAS-transformed keratinocytes, Ser260 of hRXRalpha is phosphorylated through the RAS-RAF-MAP kinase cascade. This phosphorylation event results in the inhibition of vitamin D signaling via VDR/hRXRalpha heterodimers. Strategies to reverse this resistance include the use of the MAP kinase inhibitor, PD098059, and a non-phosphorylatable hRXRalpha mutant, Ala260, which completely abolishes RXR phosphorylation and restores the function of both 1,25(OH)(2)D(3) and a specific RXR ligand, LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphtalenyl)ethenyl]-benzoic acid). In addition, we show that a vitamin D analog with low calcemic activity (EB1089) is more potent than 1,25(OH)(2)D(3) in inhibiting cancer cell growth in this system. Targeted therapy with selective analogs such as EB1089, in combination with the inhibition of phosphorylation of the RXR, could play a critical role in the development of strategies for cancer treatment.

Vitamin D Effects on Differentiation and Cell Cycle

“…It is possible that ERK facilitates the early, proliferative phase of differentiation [9,11,12,117], thus increasing the numbers of differentiated cells, rather than being related to the expression of the differentiated phenotype. This is consistent with the observation that the MEK/ERK inhibitor PD98059 substantially reduces, but does not totally prevent, 1,25(OH) 2 D 3 -induced differentiation (e.g., [13]), and the report that transformation of immortalized keratinocytes with Ha-Ras oncogene, an upstream regulator of the MEK/ERK pathway, results in resistance to the anti-proliferative action of 1,25(OH) 2 D 3 [118].…”

Phosphorylation of the Human Retinoid X Receptor α at Serine 260 Impairs Coactivator(s) Recruitment and Induces Hormone Resistance to Multiple Ligands

“…The other potential MAPK consensus sequence located around threonine 82 (9) was not phosphorylated indicating that serine 260 was likely the major site of phosphorylation of the Ras-Raf-MAPK cascade in this model. Our studies also indicated that the Ras-Raf MAPK cascade also affected the response to LG1069, a specific RXR␣ ligand (9,20), raising the possibility that serine 260 phosphorylation of RXR␣ could also affect other partners of the RXR␣ such as RXR␣⅐RXR␣, RXR␣/RAR␣, RXR␣/ TR␤, and RXR␣/PPAR␥. Indeed, subsequent studies by MatsushimaNishiwaki et al (10) indicated that vitamin A signaling through RAR␣⅐ RXR␣ was also impaired by MAPKdependent phosphorylation at serine 260 in hepatocellular carcinoma.…”

Phosphorylation of retinoid X receptor suppresses its ubiquitination in human hepatocellular carcinoma

“…In addition, phosphoserine 260 RXR␣ has been reported to interfere with vitamin D3 receptor/ RXR-dependent signaling, and thereby confers a hormone-resistant growth advantage in ras-transformed human keratinocytes. 34,35 Thus, phosphorylation of RXR␣ might well be linked to the carcinogenic process by impairing its function as well as by accumulating its inactive or nontransactivating receptor, resulting in the failure to regulate cell proliferation.…”