Metabolic Reprogramming by Ribitol Expands the Therapeutic Window of BETi JQ1 against Breast Cancer.

Many cancer patients still lack effective treatments, and pre-existing or acquired resistance limits the clinical benefit of even the most advanced medicines. Recently, much attention has been given to the role of metabolism in cancer, expanding from the Warburg effect to highlight unique patterns that, in turn, may improve diagnostic and therapeutic approaches. Our recent metabolomics study revealed that ribitol can alter glycolysis in breast cancer cells. In the current study, we investigate the combinatorial effects of ribitol with several other anticancer drugs (chrysin, lonidamine, GSK2837808A, CB-839, JQ1, and shikonin) in various breast cancer cells (MDA-MB-231, MCF-7, and T-47D). The combination of ribitol with JQ1 synergistically inhibited the proliferation and migration of breast cancer cells cell-type dependently, only observed in the triple-negative MDA-MB-231 breast cancer cells. This synergy is associated with the differential effects of the 2 compounds on expression of the genes involved in cell survival and death, specifically downregulation in c-Myc and other anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1), but upregulation in p53 and cytochrome C levels. Glycolysis is differentially altered, with significant downregulation of glucose-6-phosphate and lactate by ribitol and JQ1, respectively. The overall effect of the combined treatment on metabolism and apoptosis-related genes results in significant synergy in the inhibition of cell growth and induction of apoptosis. Given the fact that ribitol is a metabolite with limited side effects, a combined therapy is highly desirable with relative ease to apply in the clinic for treating an appropriate cancer population. Our results also emphasize that, similar to traditional drug development, the therapeutic potential of targeting metabolism for cancer treatment may only be achieved in combination with other drugs and requires the identification of a specific cancer population. The desire to apply metabolomic intervention to a large scope of cancer types may be one of the reasons identification of this class of drugs in a clinical trial setting has been delayed.

Ribitol enhances matriglycan of α-dystroglycan in breast cancer cells without affecting cell growth.

The laminin-binding glycan (matriglycan) on α-dystroglycan (α-DG) enables diverse roles, from neuronal development to muscle integrity. Reduction or loss of matriglycan has also been implicated in cancer development and metastasis, and specifically associated with high-grade tumors and poor prognoses in breast cancers. Hyperglycosylation of α-DG with LARGE overexpression is shown to inhibit cancer cell growth and tumorigenicity. We recently demonstrated that ribitol, considered to be a metabolic end-product, enhances matriglycan expression in dystrophic muscles in vivo. In the current study, we tested the hypothesis that ribitol could also enhance matriglycan expression in cancer cells. Our results showed for the first time that ribitol is able to significantly enhance the expression of matriglycan on α-DG in breast cancer cells. The ribitol effect is associated with an increase in levels of CDP-ribitol, the substrate for the ribitol-5-phosphate transferases FKRP and FKTN. Direct use of CDP-ribitol is also effective for matriglycan expression. Ribitol treatment does not alter the expression of FKRP, FKTN as well as LARGEs and ISPD which are critical for the synthesis of matriglycan. The results suggest that alteration in substrates could also be involved in regulation of matriglycan expression. Interestingly, expression of matriglycan is related to cell cycle progression with highest levels in S and G2 phases and ribitol treatment does not alter the pattern. Although matriglycan up-regulation does not affect cell cycle progression and proliferation of the cancer cells tested, the novel substrate-mediated treatment opens a new approach easily applicable to experimental systems in vivo for further exploitation of matriglycan expression in cancer progression and for therapeutic potential.

Overexpression of Mutant FKRP Restores Functional Glycosylation and Improves Dystrophic Phenotype in FKRP Mutant Mice.

Autosomal recessive homozygous or compound heterozygous mutations in FKRP result in forms of muscular dystrophy-dystroglycanopathy varying in age of onset, clinical presentation, and disease progression, ranging from the severe Walker-Warburg, type A,5 (MDDGA5), muscle-eye-brain (MDDGB5) with or without cognitive deficit, to limb-girdle type 2I (MDDGC5). Phenotypic variation indicates degrees of functionality of individual FKRP mutation, which has been supported by the presence of residual expression of functionally glycosylated α-dystroglycan (DG) in muscles of both animal models and patients. However, direct evidence showing enhancement in glycosylation of α-DG by mutant FKRP is lacking. Using AAV9-mediated overexpression of mutant human FKRP bearing the P448L mutation (mhFKRP-P448L) associated with severe congenital muscular dystrophy (CMD), we demonstrate the restoration of functional glycosylation of α-DG and reduction in markers of disease progression. Expression of mhFKRP-P448L also corrects dystrophic phenotypes in the models of L276I mutation with mild disease phenotype and causes no obvious histological or biomarker alteration in C57BL/6 normal mice. Our results confirm the existing function of mutant FKRP. The results also suggest that mutant FKRP could be an alternative approach for potential gene therapy should normal FKRP gene products be immunogenic.

Expression of glycosylated α-dystroglycan in newborn skeletal and cardiac muscles of fukutin related protein (FKRP) mutant mice.

INTRODUCTION: Mutations in the Fukutin related protein (FKRP) gene are characterized by a lack of functionally glycosylated α-dystroglycan (F-α-DG) in muscles. A small number of fibers retain the capacity to produce strong IIH6 reactive glycosylated-α-DG (g-α-DG) in muscles of both FKRP mutant animals and patients. METHODS: We examined the expression of g-α-DG in limb, diaphragm, and cardiac muscles of newborn FKRP mutants and LARGEmyd mice with IIH6 antibody. RESULTS: Near-normal levels of g-α-DG were detected in all 3 muscles in the FKRP448LNeo- mutant. Expression was limited within the first 8 postnatal days with decreasing levels. No expression was identified in LARGEmyd mice. CONCLUSIONS: Temporary expression of glycosylated-α-DG in newborn FKRP mutant muscles is LARGE- and mutant FKRP-dependent. The capability of mutant FKRP with a severe clinic phenotype to produce glycosylated-α-DG provides a new perspective for possible approaches to mitigate FKRP deficiency. Muscle Nerve 55: 582-590, 2017.

Mutations alter secretion of fukutin-related protein.

Mutations in the fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker-Warburg syndrome, muscle-eye-brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-related diseases.

Fukutin-related protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo.

Mutations in the fukutin-related protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-related myopathies.