Flavopiridol Suppresses Cell Proliferation and Migration and Induces Apoptotic Cell Death by Inhibiting Oncogenic FOXM1 Signaling in IDH Wild-Type and IDH-Mutant GBM Cells.

Glioblastoma multiforme (GBM) remains one of the most challenging solid cancers to treat due to its highly aggressive and drug-resistant nature. Flavopiridol is synthetic flavone that was recently approved by the FDA for the treatment of acute myeloid leukemia. Flavopiridol exhibits antiproliferative activity in several solid cancer cells and currently evaluated in clinical trials in several solid and hematological cancers. In this study, we investigated the molecular mechanisms underlying antiproliferative effects of flavopiridol in GBM cell lines with wild-type and mutant encoding isocitrate dehydrogenase 1 (IDH1). We found that flavopiridol inhibits proliferation, colony formation, and migration and induces apoptosis in IDH1 wild-type and IDH-mutant cells through inhibition of FOXM1 oncogenic signaling. Furthermore, flavopiridol treatment also inhibits of NF-KB, mediators unfolded protein response (UPR), including, GRP78, PERK and IRE1α, and DNA repair enzyme PARP, which have been shown to be potential therapeutic targets by downregulating FOXM1 in GBM cells. Our findings suggest for the first time that flavopiridol suppresses proliferation, survival, and migration and induces apoptosis in IDH1 wild-type and IDH1-mutant GBM cells by targeting FOXM1 oncogenic signaling which also regulates NF-KB, PARP, and UPR response in GBM cells. Flavopiridol may be a potential novel therapeutic strategy in the treatment of patients IDH1 wild-type and IDH1-mutant GBM.

The Role of Apoptosis and Autophagy in the Hypothalamic-Pituitary-Adrenal (HPA) Axis after Traumatic Brain Injury (TBI).

Traumatic brain injury (TBI) is a major health problem affecting millions of people worldwide and leading to death or permanent damage. TBI affects the hypothalamic-pituitary-adrenal (HPA) axis either by primary injury to the hypothalamic-hypophyseal region or by secondary vascular damage, brain, and/or pituitary edema, vasospasm, and inflammation. Neuroendocrine dysfunctions after TBI have been clinically described in all hypothalamic-pituitary axes. We established a mild TBI (mTBI) in rats by using the controlled cortical impact (CCI) model. The hypothalamus, pituitary, and adrenals were collected in the acute (24 h) and chronic (30 days) groups after TBI, and we investigated transcripts and protein-related autophagy (Lc3, Bcln1, P150, Ulk, and Atg5) and apoptosis (pro-caspase-3, cleaved caspase-3). Transcripts related to autophagy were reduced in the hypothalamus, pituitary, and adrenals after TBI, however, this was not reflected in autophagy-related protein levels. In contrast, protein markers related to apoptosis increased in the adrenals during the acute phase and in the pituitary during the chronic phase. TBI stresses induce a variation of autophagy-related transcripts without modifying the levels of their proteins in the HPA axis. In contrast, protein markers related to apoptosis are increased in the acute phase in the adrenals, which could lead to impaired communication via the hypothalamus, pituitary, and adrenals. This may then explain the permanent pituitary damage with increased apoptosis and inflammation in the chronic phase. These results contribute to the elucidation of the mechanisms underlying endocrine dysfunctions such as pituitary and adrenal insufficiency that occur after TBI. Although the adrenals are not directly affected by TBI, we suggest that the role of the adrenals along with the hypothalamus and pituitary should not be ignored in the acute phase after TBI.

Serotonin 5-HT7 receptor is a biomarker poor prognostic factor and induces proliferation of triple-negative breast cancer cells through FOXM1.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer and associated with poor prognosis and shorter survival due to significant genetic heterogeneity, drug resistance and lack of effective targeted therapeutics. Therefore, novel molecular targets and therapeutic strategies are needed to improve patient survival. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce growth stimulatory effects in breast cancer. However, the molecular mechanisms by which 5-HT exerts its oncogenic effects in TNBC still are not well understood. METHODS: Normal breast epithelium (MCF10A) and two TNBC cells (MDA-MB-231, BT-546) and MCF-7 cells (ER +) were used to investigate effects of 5-HT7 receptor. Small interfering RNA (siRNA)-based knockdown and metergoline (5-HT7 antagonist) were used to inhibit the activity of 5-HT7. Cell proliferation and colony formation were evaluated using MTS cell viability and colony formation assays, respectively. Western blotting was used to investigate 5-HT7, FOXM1 and its downstream targets protein expressions. RESULTS: We demonstrated that 5-HT induces cell proliferation of TNBC cells and expression of 5-HT7 receptor and FOXM1 oncogenic transcription factor. We found that expression of 5-HT7 receptor is up-regulated in TNBC cells and higher 5-HT7 receptor expression is associated with poor patient prognosis and shorter patient survival. Genetic and pharmacological inhibition of 5-HT7 receptor by siRNA and metergoline, respectively, suppressed TNBC cell proliferation and FOXM1 and its downstream mediators, including eEF2-Kinase (eEF2K) and cyclin-D1. CONCLUSION: Our findings suggest for the first time that the 5-HT7 receptor promotes FOXM1, eEF2K and cyclin D1 signaling to support TNBC cell proliferation; thus, inhibition of 5-HT7 receptor/FOXM1 signaling may be used as a potential therapeutic strategy for targeting TNBC. 5-HT induces cell proliferation of TNBC cells through 5-HT7 receptor signaling. Also, genetic and pharmacological inhibition of 5-HT7 by RNAi (siRNA) and metergoline HTR7 antagonist, respectively inhibits FOXM1 oncogenic transcription factor and suppresses TNBC cell proliferation.

MicroRNA profiling identifies Forkhead box transcription factor M1 (FOXM1) regulated miR-186 and miR-200b alterations in triple negative breast cancer.

Breast cancer (BC) is the most commonly diagnosed malignancy. MicroRNAs (miRNAs) play important roles in the tumorigenesis, metastasis and progression of BC. Forkhead Box M1 (FOXM1) oncogenic transcription factor is involved in events considered as hallmarks of cancer. However, the specific mechanism by which FOXM1 exerts its oncogenic effects remains unclear and little is known about its effects on the regulation of miRNA expression. We have found that FOXM1 is upregulated in breast cancer cells and that its expression is associated with shortened overall survival and poor prognosis in patients with BC. Using microarray technology, we assessed the expression profiles of 752 miRNAs in highly aggressive and metastatic triple negative breast cancer (TNBC) cells in response to FOXM1 knockdown and identified 13 differentialy expressed miRNAs (3 miRNAs upregulated and 10 miRNAs down-regulated). We validated the results of the miRNA expression profile in two different TNBC cells by performing qRT-PCR and identified that miR-186-5p and miR-200b-5p were consistently down- or up-regulated, respectively, after knockdown of FOXM1. We further performed KEGG pathway analysis and GO enrichment analysis for miR-186-5p and miR-200b-5p, and identified that these miRNAs are associated with cancer development and progression involving toll-like receptor signaling, cell cycle, AMPK, p53 and NF-kappa B signaling pathways. Taken together, our results suggest that increased FOXM1 expression is associated with poor patient survival and leads to induction of oncomiR miR-186-5p expression and tumor-suppressor inhibition miR-200b-5p, suggesting that the FOXM1/miRNA signaling pathway may contribute to poor patient prognosis and may be a potential therapeutic target in TNBC.

Thymoquinone Inhibits Proliferation and Migration of MDA-MB-231 Triple Negative Breast Cancer Cells by Suppressing Autophagy, Beclin-1 and LC3.

BACKGROUND: Triple Negative Breast Cancer (TNBC) is an aggressive and highly heterogeneous subtype of breast cancer associated with poor prognosis. A better understanding of the biology of this complex cancer is needed to develop novel therapeutic strategies for the improvement of patient survival. We have previously demonstrated that Thymoquinone (TQ), the major phenolic compound found in Nigella sativa, induces anti-proliferative and anti-metastatic effects and inhibits in vivo tumor growth in orthotopic TNBC models in mice. Also, we have previously shown that Beclin-1 and LC3 autophagy genes contributes to TNBC cell proliferation, migration and invasion, suggesting that Beclin-1 and LC3 genes provide proto-oncogenic effects in TNBC. However, the role of Beclin-1 and LC3 in mediating TQ-induced anti-tumor effects in TNBC is not known. OBJECTIVE: To investigate the effects of TQ on the major autophagy mediators, Beclin-1 and LC3 expression, as well as autophagic activity in TNBC cells. METHODS: Cell proliferation, colony formation, migration and autophagy activity were evaluated using MTS cell viability, colony formation assay, wound healing and acridine orange staining assays, respectively. Western blotting and RT-PCR assays were used to investigate LC3 and Beclin-1 protein and gene expressions, respectively, in MDA-MB-231 TNBC cells in response to TQ treatments. RESULTS: TQ treatment significantly inhibited cell proliferation, colony formation, migration and autophagic activity of MDA-MB-231 cells and suppressed LC3 and Beclin-1 expressions. Furthermore, TQ treatment led to the inhibition of Integrin-ß1, VEGF, MMP-2 and MMP-9 in TNBC cells. CONCLUSION: TQ inhibits autophagic activity and expression of Beclin-1 and LC3 in TNBC cells and suppresses pathways related to cell migration/invasion and angiogenesis, including Integrin-ß1, VEGF, MMP-2 and MMP- 9, suggesting that TQ may be used to control autophagic activity and oncogenic signaling in TNBC.