Parallel to complex alteration in molecular and cellular events, enrichment of cancer stem cells (CSC) contributes significantly in deliberation and maintenance of cisplatin resistance. Cisplatin mediated CSC enrichment is well established in various cancers, yet the underlying mechanism is largely unknown. Cisplatin also promotes transcriptional upregulation of PIK3CA, hence activating PI3K/AKT signaling in resistant cells. However, such cisplatin-induced transcriptional regulators of PIK3CA and their impact on cancer stem cell population in resistant cells are largely unknown. DNA-binding protein pulldown using PIK3CA promoter as bait followed by nLCMS was used to identify, cisplatin-induced potential transcriptional regulators of PIK3CA promoter. PIK3CA promoter activity was estimated by luciferase based reporter assay. ChIP was used to assess interaction of NF-κB with PIK3CA promoter. CSC-enriched side-population was sorted using DCV-dye exclusion methods. All the gene expression levels were assessed using qPCR. Using a transcription factor pull-down assay with PIK3CA promoter, we identified NF-κB as a prime regulator, which escalates both TNFα and PIK3CA expression only in CSC enriched side-population (SP) but not in non side-population (NSP) in platinum resistant ovarian cancer cells upon cisplatin treatment. This SP-specific NF-κB-TNFα-PIK3CA bi-modal loop, on one hand, maintains persistent activation of NF-κB through TNFα- NF-κB autocrine loop, while NF-κB-PIK3CA loop nurture CSC population under cisplatin treatment. Activation of PI3K/AKT signalling drives SP’s into an undifferentiated, anti-apoptotic stage through upregulating P21, P27,cFLIP expression. Contrarily, lack of active NF-κB-TNFα-PIK3CA loop makes NSPs vulnerable towards cisplatin and undergoes apoptosis. Altogether, cisplatin enriches cancer stem cells properties in SP fraction, which is evident from increased levels of pluripotency gene OCT4/SOX2/NANOG expression. Disruption of PIK3CA-NF-κB loop by Wortamannin reduces SP fraction by 1.4–1.6 fold in control and treated cells. Together, our study signifies an active role of NF-κB-TNFα-PIK3CA bi-modal loop in cisplatin-mediated promotion and maintenance of CSC-like population in platinum-resistant cells.

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin–RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.