Molecular and Cellular Biology

Nuclear Lamin A/C Expression Is a Key Determinant of Paclitaxel Sensitivity

Paclitaxel is a key member of the Taxane (paclitaxel [originally named taxol], docetaxel/Taxotere) family of successful drugs used in the current treatment of several solid tumors, including ovarian cancer. The molecular target of paclitaxel has been identified as tubulin, and paclitaxel binding alters the dynamics and thus stabilizes microtubule bundles. Traditionally, the anticancer mechanism of paclitaxel has been thought to originate from its interfering with the role of microtubules in mitosis, resulting in mitotic arrest and subsequent apoptosis. However, recent evidence suggests that paclitaxel operates in cancer therapies via an as-yet-undefined mechanism rather than as a mitotic inhibitor. We found that paclitaxel caused a striking break up of nuclei (referred to as multimicronucleation) in malignant ovarian cancer cells but not in normal cells, and susceptibility to undergo nuclear fragmentation and cell death correlated with a reduction in nuclear lamina proteins, lamin A/C. Lamin A/C proteins are commonly lost, reduced, or heterogeneously expressed in ovarian cancer, accounting for the aberration of nuclear shape in malignant cells. Mouse ovarian epithelial cells isolated from lamin A/C-null mice were highly sensitive to paclitaxel and underwent nuclear breakage, compared to control wild-type cells. Forced overexpression of lamin A/C led to resistance to paclitaxel-induced nuclear breakage in cancer cells. Additionally, paclitaxel-induced multimicronucleation occurred independently of cell division that was achieved by either the withdrawal of serum or the addition of mitotic inhibitors. These results provide a new understanding for the mitotis-independent mechanism for paclitaxel killing of cancer cells, where paclitaxel induces nuclear breakage in malignant cancer cells that have a malleable nucleus but not in normal cells that have a stiffer nuclear envelope. As such, we identify that reduced nuclear lamin A/C protein levels correlate with nuclear shape deformation and are a key determinant of paclitaxel sensitivity of cancer cells.

LATS1 Regulates Mixed-Lineage Kinase 3 (MLK3) Subcellular Localization and MLK3-Mediated Invasion in Ovarian Epithelial Cells

Mixed-lineage kinase 3 (MLK3) activates mammalian mitogen-activated protein kinase (MAPK) signaling pathways in response to cytokines and stress stimuli. MLK3 is important for proliferation, migration, and invasion of different types of human tumor cells. We observed that endogenous MLK3 was localized to both the cytoplasm and the nucleus in immortalized ovarian epithelial (T80) and ovarian cancer cells, and mutation of arginines 474 and 475 within a putative MLK3 nuclear localization sequence (NLS) resulted in exclusion of MLK3 from the nucleus. The large tumor suppressor (LATS) Ser/Thr kinase regulates cell proliferation, morphology, apoptosis, and mitotic exit in response to cell-cell contact. RNA interference (RNAi)-mediated knockdown of LATS1 increased nuclear, endogenous MLK3 in T80 cells. LATS1 phosphorylated MLK3 on Thr477, which is within the putative NLS, and LATS1 expression enhanced the association between MLK3 and the adapter protein 14-3-3ζ. Thr477 is essential for MLK3-14-3-3ζ association and MLK3 retention in the cytoplasm, and a T477A MLK3 mutant had predominantly nuclear localization and significantly increased invasiveness of SKOV3 ovarian cancer cells. This study identified a novel link between the MAPK and Hippo/LATS1 signaling pathways. Our results reveal LATS1 as a novel regulator of MLK3 that controls MLK3 nuclear/cytoplasmic localization and MLK3-dependent ovarian cancer cell invasion.

LINC00997/MicroRNA 574-3p/CUL2 Promotes Cervical Cancer Development via Mitogen-Activated Protein Kinase Signaling

Cervical cancer (CC) is a common gynecological malignancy with high morbidity and mortality. Mounting evidence has highlighted that long noncoding RNAs are essential regulators in cancer development. Long intergenic non-protein-coding RNA 997 (LINC00997) was identified for study due to its high expression in CC tissues. The aim of the study was to investigate the function and mechanism of LINC00997 in CC. Reverse transcription-quantitative PCR (RT-qPCR) revealed that LINC00997 RNA expression was also increased in CC cells and LINC00997 copy number was upregulated in CC tissues. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), colony formation, and Transwell assays as well as transmission electron microscopy observation exhibited that LINC00997 depletion inhibited CC cell proliferation, migration, invasion, and autophagy. The relationship between LINC00997 and its downstream genes was confirmed by RNA pulldown, luciferase reporter, and RNA-binding protein immunoprecipitation assays. Mechanistically, LINC00997 upregulated the expression of cullin 2 (CUL2) by interacting with microRNA 574-3p (miR-574-3p). Moreover, Western blot analysis was employed to detect the protein levels of mitogen-activated protein kinase (MAPK) pathway-associated factors in CC cells. LINC00997 activated the MAPK signaling by increasing CUL2 expression, thus promoting malignant phenotypes of CC cells. In conclusion, the LINC00997/miR-574-3p/CUL2 axis contributes to CC cell proliferation, migration, invasion, and autophagy via the activation of MAPK signaling.

HNRNPU-AS1 Regulates Cell Proliferation and Apoptosis via the MicroRNA 205-5p/AXIN2 Axis and Wnt/β-Catenin Signaling Pathway in Cervical Cancer

Long noncoding RNAs (lncRNAs) have key functions in modulating cervical cancer (CC) genesis and progression. This work focused on exploring lncRNA HNRNPU-AS1's function in CC and the underlying mechanism. HNRNPU-AS1, AXIN2, and microRNA 205-5p (miR-205-5p) levels in CC cases were measured through reverse transcription-quantitative PCR. The relationship between miR-205-5p and AXIN2 or HNRNPU-AS1 was validated through a dual-luciferase assay. Cell proliferation was examined by CCK-8 and cell apoptosis by colony formation and flow cytometry analysis. HNRNPU-AS1 expression loss could be observed in CC patients and cell lines, which predicted the dismal prognosis of CC cases. Moreover, it was identified that the miR-205-5p level was upregulated, which acted as an inhibitory target of HNRNPU-AS1 and AXIN2. HNRNPU-AS1 inhibited cell proliferation and promoted apoptosis. As revealed by Kaplan-Meier curve, CC cases showing low HNRNPU-AS1, high miR-205-5p, and low AXIN2 levels had the poorest prognosis. AXIN2 reversed the CC cell proliferation-promoting, apoptosis-inhibiting, and Wnt/β-catenin signaling-activating behavior mediated by miR-205-5p or HNRNPU-AS1 knockout. In conclusion, the overexpression of lncRNA HNRNPU-AS1 suppressed CC progression by inhibiting the Wnt/β-catenin pathway through the miR-205-5p/AXIN2 axis.

Overexpression of MicroRNA 142-5p Suppresses the Progression of Cervical Cancer through Targeting Phosphoinositol-3-Kinase Adaptor Protein 1 Expression

The aim of current study was to explore the mechanism of microRNA 142-5p (miR-142-5p) in cervical cancer through mediating the phosphoinositol-3-kinase adaptor protein 1 (PIK3AP1)/PI3K/AKT axis. To this end, reverse transcription-quantitative PCR (RT-qPCR) and Western blot analysis results revealed that miR-142-5p was poorly expressed, whereas PIK3AP1 was highly expressed, in cervical cancer tissues and cells. Furthermore, miR-142-5p was hypermethylated in cervical cancer, as reflected by methylation-specific PCR (MS-PCR) and chromatin immunoprecipitation (ChIP) assessment of enrichment of DNMT1/DNMT3a/DNMT3b in the promoter region of miR-142-5p. A target binding relationship between miR-142-5p and PIK3AP1 was established, showing that miR-142-5p targeted and inhibited the expression of PIK3AP1. Loss- and gain-of-function assays were conducted to determine the roles of miR-142-5p and PIK3AP1 in cervical cancer cells. CCK-8, flow cytometry, and Transwell assay results revealed that overexpression of miR-142-5p in cervical cancer cells downregulated PIK3AP1 and inhibited the PI3K/AKT signaling pathway, leading to reduced proliferation, migration, and invasion capacity of cervical cancer cells but enhanced apoptosis. Collectively, epigenetic regulation of miR-142-5p targeted PIK3AP1 to inactivate the PI3K/AKT signaling pathway, thus suppressing development of cervical cancer, which presents new targets for the treatment of cervical cancer.

Exosome-transmitted circ IFNGR2 Modulates Ovarian Cancer Metastasis via miR-378/ST5 Axis

Cancer-associated fibroblasts (CAFs)-derived exosomes have emerged as a key driver of ovarian cancer (OVCA) tumor progression. The mechanisms behind the specific circular RNA (circRNA) activity encapsulated by CAF-generated exosomes (CAF-exo) requires to be elucidated. Herein, this study selected specific circRNA (hsa_circ

Silencing of hsa_circ_0009035 Suppresses Cervical Cancer Progression and Enhances Radiosensitivity through MicroRNA 889-3p-Dependent Regulation of HOXB7

Circular RNAs (circRNAs), a novel type of endogenous noncoding RNAs, have been identified as critical regulators in human carcinogenesis. Here, we investigated the precise actions of hsa_circ_0009035 in the progression and radioresistance of cervical cancer (CC). The levels of hsa_circ_0009035, microRNA 889-3p (miR-889-3p), and homeobox B7 (HOXB7) were detected by quantitative real-time PCR (qRT-PCR) or Western blotting. RNase R and actinomycin D assays were used to assess the stability of hsa_circ_0009035. Cell proliferation, cell cycle progression, apoptosis, migration, and invasion were gauged with Cell Counting Kit-8 (CCK-8), flow cytometry, and transwell assays. Cell colony formation and survival were determined by the colony formation assay. Targeted correlations among hsa_circ_0009035, miR-889-3p, and HOXB7 were examined by the dual-luciferase reporter, RNA immunoprecipitation (RIP), or RNA pulldown assay. Animal studies were performed to evaluate the impact of hsa_circ_0009035 on tumor growth. We found that hsa_circ_0009035 was highly expressed in CC tissues and cells, and it was associated with the radioresistance of CC patients. Moreover, the silencing of hsa_circ_0009035 inhibited CC cell proliferation, migration, invasion, and it enhanced apoptosis and radiosensitivity