Tamoxifen Treatment of Breast Cancer Cells: Impact on Hedgehog/GLI1 Signaling.

The selective estrogen receptor (ER) modulator tamoxifen (TAM) has become the standard therapy for the treatment of ER+ breast cancer patients. Despite the obvious benefits of TAM, a proportion of patients acquire resistance to treatment, and this is a significant clinical problem. Consequently, the identification of possible mechanisms involved in TAM-resistance should help the development of new therapeutic targets. In this study, we present in vitro data using a panel of different breast cancer cell lines and demonstrate the modulatory effect of TAM on cellular proliferation and expression of Hedgehog signaling components, including the terminal effector of the pathway, the transcription factor GLI1. A variable pattern of expression following TAM administration was observed, reflecting the distinctive properties of the ER+ and ER- cell lines analyzed. Remarkably, the TAM-induced increase in the proliferation of the ER+ ZR-75-1 and BT474 cells parallels a sustained upregulation of GLI1 expression and its translocation to the nucleus. These findings, implicating a TAM-GLI1 signaling cross-talk, could ultimately be exploited not only as a means for novel prognostication markers but also in efforts to effectively target breast cancer subtypes.

Tamoxifen Resistance: Emerging Molecular Targets.

17ß-Estradiol (E2) plays a pivotal role in the development and progression of breast cancer. As a result, blockade of the E2 signal through either tamoxifen (TAM) or aromatase inhibitors is an important therapeutic strategy to treat or prevent estrogen receptor (ER) positive breast cancer. However, resistance to TAM is the major obstacle in endocrine therapy. This resistance occurs either de novo or is acquired after an initial beneficial response. The underlying mechanisms for TAM resistance are probably multifactorial and remain largely unknown. Considering that breast cancer is a very heterogeneous disease and patients respond differently to treatment, the molecular analysis of TAM's biological activity could provide the necessary framework to understand the complex effects of this drug in target cells. Moreover, this could explain, at least in part, the development of resistance and indicate an optimal therapeutic option. This review highlights the implications of TAM in breast cancer as well as the role of receptors/signal pathways recently suggested to be involved in the development of TAM resistance. G protein-coupled estrogen receptor, Androgen Receptor and Hedgehog signaling pathways are emerging as novel therapeutic targets and prognostic indicators for breast cancer, based on their ability to mediate estrogenic signaling in ERα-positive or -negative breast cancer.

Neighboring gene regulation by antisense long non-coding RNAs.

Antisense transcription, considered until recently as transcriptional noise, is a very common phenomenon in human and eukaryotic transcriptomes, operating in two ways based on whether the antisense RNA acts in cis or in trans. This process can generate long non-coding RNAs (lncRNAs), one of the most diverse classes of cellular transcripts, which have demonstrated multifunctional roles in fundamental biological processes, including embryonic pluripotency, differentiation and development. Antisense lncRNAs have been shown to control nearly every level of gene regulation--pretranscriptional, transcriptional and posttranscriptional--through DNA-RNA, RNA-RNA or protein-RNA interactions. This review is centered on functional studies of antisense lncRNA-mediated regulation of neighboring gene expression. Specifically, it addresses how these transcripts interact with other biological molecules, nucleic acids and proteins, to regulate gene expression through chromatin remodeling at the pretranscriptional level and modulation of transcriptional and post-transcriptional processes by altering the sense mRNA structure or the cellular compartmental distribution, either in the nucleus or the cytoplasm.

The impact of S6K1 kinase on neuroblastoma cell proliferation is independent of GLI1 signaling.

BACKGROUND: The crosstalk between Hedgehog (HH) signaling and other signal transduction cascades has been extensively studied in different cancers. In neuroblastoma, mTOR/S6K1 signaling is known to have a role in the development of this disease and recent evidence also implicates the HH pathway. Moreover, S6K1 kinase has been shown to phosphorylate GLI1, the effector of HH signaling, promoting GLI1 transcriptional activity and oncogenic function in esophageal adenocarcinoma. In this study, we examined the possible interplay of S6K1 and GLI1 signaling in neuroblastoma. METHODS: siRNA knockdowns were used to suppress S6K1 and GLI1 expression, and the siRNA effects were validated by real-time PCR and Western blotting. Cell proliferation analysis was performed with the EdU incorporation assay. Cytotoxic analysis with increasing concentrations of PI3K/mTOR and GLI inhibitors, individually and in combination, was used to determine drug response. RESULTS: Although knockdown of either S6K1 or GLI1 reduces the cellular proliferation of neuroblastoma cells, there is little effect of S6K1 on the expression of GLI1 mRNA and protein and on the capacity of GLI1 to activate target genes. No detectable phosphorylation of GLI1 is observed prior or following S6K1 knockdown. GLI1 overexpression can not rescue the reduced proliferation elicited by S6K1 knockdown. Moreover, inhibitors of PI3K/mTOR and GLI signaling reduced neuroblastoma cell growth, but no additional growth inhibitory effects were detected when the two classes of drugs were combined. CONCLUSION: Our results demonstrate that the impact of S6K1 kinase on neuroblastoma cells is not mediated through modulation of GLI1 expression/activity.

KRAS Promotes GLI2-Dependent Transcription during Pancreatic Carcinogenesis.

Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.

Targeting the hedgehog signal transduction pathway at the level of GLI inhibits neuroblastoma cell growth in vitro and in vivo.

Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.

RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling.

The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway.

Circular and Fusion RNAs in Medulloblastoma Development.

Background. The cerebellar cancer medulloblastoma is the most common childhood cancer in the brain. Methods. RNA sequencing of 81 human biospecimens of medulloblastoma using pipelines to detect circular and fusion RNAs. Validation via PCR and Sanger sequencing. Results. 27, 56, 28 and 11 RNA circles were found to be uniquely up-regulated, while 149, 7, 20 and 15 uniquely down-regulated in the SHH, WNT, Group 3, and Group 4 medulloblastoma subtypes, respectively. Moreover, linear and circular fusion RNAs containing exons from distinct genes joined at canonical splice sites were also identified. These were generally expressed less than the circular RNAs, however the expression of both the linear and the circular fusions was comparable. Importantly, the expression of the fusions in medulloblastoma was also comparable to that of cerebellum. Conclusions. A significant number of fusions in tumor may be generated by mechanisms similar to the ones generating fusions in normal tissue. Some fusions could be rationalized by read-through transcription of two neighboring genes. However, for other fusions, e.g., a linear fusion with an exon from a downstream gene joined 5' to 3' with an exon from an upstream gene, more complicated splicing mechanisms, e.g., trans-splicing, have to be postulated.

Circular RNAs in Hedgehog Signaling Activation and Hedgehog-Mediated Medulloblastoma Tumors.

Within the past decade, circular RNAs have largely emerged as novel regulators of human biology, including brain function and cancer development. On the other hand, the Hedgehog pathway has established roles in regulating biological processes, including tumorigenesis. Here, the circular RNA transcriptome, in the context of Hedgehog signaling activation of medulloblastoma Daoy and human embryonic palatal mesenchyme HEPM cells, was determined. In total, 29 out of the 30 selected circular RNAs were validated by Sanger sequencing, with some regulated to a limited extent by Hedgehog signaling. Interestingly, back-spliced junctions, the marker of exonic RNA circles, were also identified at a low frequency within poly (A) mRNAs, reflecting exon repetition events. Thirteen circular RNAs had reduced expression in human medulloblastoma tumors in comparison to normal cerebellum. For seven out of these thirteen RNA circles, the linear mRNAs originating from the same genes did not exhibit a reduced expression. Depletion and/or overexpression of these seven circular RNAs minimally affected medulloblastoma cell proliferation. These findings highlight that differential expression of a gene product may not necessarily elicit an obvious phenotypic impact. Consequently, further analysis is required to determine the possible subtle contributions to the development of this cerebellar tumor.

Genetic variations regulate alternative splicing in the 5' untranslated regions of the mouse glioma-associated oncogene 1, Gli1.

BACKGROUND: Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs) of mRNAs, the understanding of the significance and the regulation of these variations is rather limited. RESULTS: We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH) signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective. CONCLUSIONS: Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.