The c-Raf modulator RRD-251 enhances nuclear c-Raf/GSK-3/VDR axis signaling and augments 1,25-dihydroxyvitamin D3-induced differentiation of HL-60 myeloblastic leukemia cells

Abstract

Differentiation therapy is used in cancer treatment. Epidemiologic studies showed that higher vitamin D levels are associated with reduced cancer risks. However, the therapeutic doses needed for differentiation are accompanied by hypercalcemia and intolerable pathological sequelae. In the present work we evaluated if RRD-251, a small-molecule, can enhance vitamin D3-induced differentiation of leukemic cells, in the hope of decreasing the needed vitamin D3-dose.We demonstrate that RRD-251 enhances vitamin D3-induced differentiation of leukemic cells, the enrichment of the c-Raf kinase in the nucleus, the binding of nuclear c-Raf to GSK-3, increased phosphorylation of GSK-3 ser 21/9 inhibitory sites, and the binding of GSK-3 to VDR, where GSK-3 inhibition is known to enhance transcriptional activation by VDR. Enhancement of D3-induced p-GSK-3 ser 21/9 by RRD-251 was associated with enhanced Akt-GSK-3 binding, Akt being a known GSK-3 inhibitor, and diminished Erk1/2 binding. Diminishing Erk interaction with GSK-3 was associated with enhanced interaction with Vav1, a known driver of myeloid differentiation. This is redolent of an ATRA/c-Raf/GSK-3/RARα axis we previously reported, although the phosphorylation effects to enhance transcriptional activation on RARα vs VDR diverge. Taken together this indicates potential therapeutic significance for a c-Raf/GSK-3/VDR or RARα axis for leukemic myelo-monocytic differentiation.

Roscovitine enhances all-trans retinoic acid (ATRA)-induced nuclear enrichment of an ensemble of activated signaling molecules and augments ATRA-induced myeloid cell differentiation

“…We analyzed the expression of total RB and pS608-RB status in these cells. Consistent with other reports [14, 16,39,[45][46][47], ATRA promoted the down-regulation of total RB and diminished the amount of pS608-RB, and co-treatment with roscovitine enhanced this ( Figure 6F and 6G). Thus, roscovitine enhanced ATRA-induced effects on G1/G0 cell cycle arrest through regulatory proteins of the p27/cyclin E1/ Cdk2/RB pathway.…”

“…ATRA induces these cells to undergo myeloid differentiation and G0 cell cycle arrest that depends on a sustained MAPK pathway signal with up-regulation and unanticipated translocation of c-Raf to the nucleus [14,15]. In the nucleus, c-Raf interacts with the RB tumor suppressor protein [14,16,17]. RB is considered a master regulator of the cell cycle that becomes progressively phosphorylated with G1/S/G2 progression.…”

Effect Modification of Vitamin D Supplementation by Histopathological Characteristics on Survival of Patients with Digestive Tract Cancer: Post Hoc Analysis of the AMATERASU Randomized Clinical Trial

“…Therefore, it is possible that vitamin D may improve survival time in patients with undifferentiated, but not differentiated carcinoma. In-vitro experiments [2,3,4] have shown the potential for vitamin D to induce differentiation of undifferentiated cancer cells in vivo. In fact, a clinical pilot trial indicated that vitamin D may increase differentiation in the normal colorectal mucosa of patients with colorectal adenoma [22], although that trial did not include patients with cancer.…”

“…The 25(OH)D is further activated in the kidneys by 1-alpha-hydroxylase to 1,25-dihydroxyvitamin D, which facilitates calcium absorption and plays an important role in bone health. A more recent discovery that most tissues, as well as cancers, have 1-alpha-hydroxylase to convert serum 25(OH)D to 1,25(OH)2D, along with vitamin D receptors, which are steroid hormone nuclear receptors that regulate a variety of genes within a cell [1], has provided new insights into the function of vitamin D. In particular, vitamin D has been demonstrated to induce differentiation of cancer cells in vitro [2,3,4], which leads to the hypothesis that vitamin D supplementation may improve prognosis of patients with poorly differentiated carcinoma in vivo.…”

“…Vitamin D has been repeatedly shown to induce differentiation of immature cancer cells in vitro [2,3,4]. In contrast, to the best of our knowledge, vitamin D has not been assessed in terms of its effects by maturity levels of carcinoma, e.g., well, moderately, and poorly differentiated carcinoma through examining pathological specimen derived from patients.…”