TITLE:
Gene Expression Profiling of Human c-Kit Mutant D816V
AUTHORS:
Shilpa Sharma, Gurudutta Gangenahalli
KEYWORDS:
c-Kit Mutant, Hematopoietic Cells, Microarray Gene Expression, Proliferation
JOURNAL NAME:
Journal of Cancer Therapy,
Vol.7 No.6,
June
23,
2016
ABSTRACT: The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its
ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis
in the hematopoietic stem and progenitor cells. However, gain-of-function mutations
alter c-Kit auto-regulatory mechanisms to aberrant c-Kit signaling, leading to the
onset or progression of cancerous transformations. The most common mutation of c-Kit
is the substitution of aspartic acid residue in position 816 to valine (D816V),
which is majorly responsible for its ligand-independent constitutive activation,
and is implicated in hematopoietic malignancies. Currently, molecular targeted therapy
is increasingly becoming a hot spot due to its specificity and low toxicity. As
the molecular mechanisms responsible for D816V-c-Kit mediated tumorogenicity are
largely unknown, in this study, we aimed to investigate the D816V-c-Kit signaling
mediated downstream molecular targets. Specifically, we created c-Kit active mutant
form D816V and performed inducible gene expression of mutant D816V-c-Kit in monomyelocytic
cell line U937. Mutant D816V-c-Kit expressing cells revealed significantly enhanced
cellular mitogenic activity compared to wild-type c-Kit expressing cells independent
of huSCF. To examine the molecular targets regulating tumorogenic proliferation,
we evaluated the consequences of mutant D816V-c-Kit expression on downstream gene
expression profile by high throughput microarray technology. The levels of some
of the relevant genes (PIK3CB, eIF4B, PRKCDBP,
MOAP1) were validated by quantitative polymerase chain reaction. SLA, STAT5B,
MAP3K2 and MAPK14 emerged as important downstream molecular targets of mutant D816V-c-Kit.
Further, by dissecting the signaling pathways, we also demonstrated that the D816V-c-Kit
mediated hematopoietic cell proliferation is dependent on molecular target p38 MAP
kinase.