Oral Administration Of Attenuated S.typhimurium Carring ShRNA-expressing Vectors As A Cancer Therapeutic Fezf2, A Spatially Restricted Factor, Regulates Coordinated Neural Specification Through Feedback Regulation Of Notch Signaling

PartⅠ. Oral administration of attenuated S.typhimurium carring shRNA-expressing vectors as a cancer therapeuticRNA interference (RNAi), conserved throughout many eukaryotic organisms, is the sequence-specific, post-transcriptional gene silencing process initiated by double-stranded RNAs (dsRNAs) which are homologous to the gene being suppressed. Ever since its discovery in nematode worms, RNAi has become an extremely useful experimental tool in genomics. Moreover, RNAi has been supposed as a potential treatment for cancer. Despite its great potential, moving siRNA forward into the clinic is beset by problems with siRNA stability and delivery. Although, shRNA-expressing vectors can mediate the long-term knockdown of endogenous transcripts in cell culture and in viv, efficient delivery of the vectors still remains defective.In cancer research, efforts have been made to seek a magic bullet that could selectively target and destroy malignant cells. The presence of bacteria in human tumors recurrently attracted interests. Based on the observation that upon oral or intravenous administration, S.typhimurium are capable of both preferentially accumulating in tumors and inhibiting tumor growth, they have been exploited as a potential oncolytic agent. In addition, S.typhimurium can also be used to deliver tumoricidal molecules. Here, given the tumor-targeted capacity of S.typhimurium, we decided to explore the possibility of targeting genes in tumor cells with attenuated Salmonella carrying shRNA-expressing vectors that elicit RNAi.The prevalent target for antitumor therapy Bcl-2 is expressed in>90% of all malignant melanomas and its expression can reflect an increased malignant potential caused by an inhibition of apoptosis. Upon oral administration, S.typhimurium carrying shRNA-expressing vectors targeting bcl2 induced significant gene silencing in murine melanoma cells that led to a remarkably delayed tumor growth and prolonged survival in the mouse model. These results suggest that anti-tumor bacteria mediated RNAi may be a new and potent approach to the treatment of cancers. PartⅡ. Fezf2, a spatially restricted factor, regulates coordinated neural specification through feedback regulation of Notch signalingThe vertebrate central nervous system (CNS) is one of the most complex organs. It is composed of diverse cell types that differ in number, location, neurotransmitter synthesis, and connective pattern. How embryonic progenitors produce diverse differentiated cell types in a spatially and temporally coordinated manner is not well understood. Here, we report that the evolutionarily conserved zinc finger transcription factor Fezf2, which demarcates anterior forebrain progenitors, controls coordinated neural differentiation in the posterior tuberculum and hypothalamus (PT/H) of zebrafish, two basal forebrain structures that exhibit strong conservation across vertebrate species. Fezf2 loss-of-function causes deficits of not only dopaminergic, serotonergic and isotocinergic neurons, but also GABAergic and lslet+ interneurons in the PT/H region, and a converse increase of olig2-expressing oligodendrocyte precursor cells. We further show that the, dopaminergic, serotonergic and GABAergic neurons defective in Fezf2 loss-of-function share in common their birth timing. To control the coordinated formation of different neuronal types, Fezf2 demacrates neural progenitor domains expressing distinct proneural and lineage-specific factors including Neurogenin1, Ascl1a, Dl×2 and Otp, which are shown to be genetic targets of Fezf2. Fezf2 regulation of these proneural genes in progenitor cells activates Notch signaling, which in turn repress Fezf2 expression. Thus, Fezf2, a spatially restricted factor, promotes ordered neural differentiation through feedback regulation of Notch signaling. We propose that cross-regulation between region-specific progenitor factors (such as Fezf2 in the brain) and Notch signaling may be a general mechanism in ordered production of differentiated cell types during organogenesis.