Amit Kumar Srivastava

Tumor-associated macrophages contribute to cisplatin resistance via regulating Pol η-mediated translesion DNA synthesis in ovarian cancer

Tumor-associated macrophages (TAMs) are known to be involved in the manifestation of aggressive and therapy-resistant phenotypes in solid tumors. Nevertheless, the effects of dynamic intervention by TAMs on the DNA damage response of cancer cells are largely unexplored. Herein, we report that TAMs modulate the DNA damage repair pathways of ovarian cancer cells in response to platinum-(Pt) based therapeutic regimen. We demonstrate that coculture of TAMs with cancer cells directly upregulate Pol η, along with RAD18 and REV1 of the Translesion DNA synthesis (TLS) pathway, while concurrently downregulating components of the high-fidelity nucleotide excision repair (NER) mechanism. Consequently, we observed a better survival probability, DNA repair capacity, and enrichment of stemness properties in ovarian cancer cells. DNA bulky adducts produced by cisplatin are resolved through differential activation NER and TLS pathways. However, we elucidated that TAMs provide favorable conditions for activating the error-prone TLS pathway for lesion bypass over damage resolution. Furthermore, cellular crosstalk in cocultured cancer cells stimulates the nuclear translocation and expression of RelA, which recruits Pol η by acting as a potent transcription factor. In fact, with pristimerin-mediated disruption of p65 (RelA) translocation, the cancer cells become more prone to DNA damage-induced cell death and compromised regenerative potential. In both in vitro cell cultures and in vivo mouse xenograft models, cocultured macrophages exhibited predominantly M2-like phenotype with prevalence in the invasive zone of xenograft tumor margins. Taken together, our investigation revealed multifaceted crosstalk-mediated regulation of DNA damage repair between TAMs and ovarian cancer cells.

Neil 1 deficiency facilitates chemoresistance through upregulation of RAD18 expression in ovarian cancer stem cells

Over the past decades, cancer stem cells (CSCs) have emerged as a critical subset of tumor cells associated with tumor recurrence and resistance to chemotherapy. Understanding the mechanisms underlying CSC-mediated chemoresistance is imperative for improving cancer therapy outcomes. This study delves into the regulatory role of NEIL1, a DNA glycosylase, in chemoresistance in ovarian CSCs. We first observed a decreased expression of NEIL1 in ovarian CSCs, suggesting its potential involvement in CSC regulation. Using pan-cancer analysis, we confirmed the diminished NEIL1 expression in ovarian tumors compared to normal tissues. Furthermore, NEIL1 downregulation correlated with an increase in stemness markers and enrichment of CSCs, highlighting its role in modulating CSC phenotype. Further mechanistic investigation revealed an inverse correlation between NEIL1 and RAD18 expression in ovarian CSCs. NEIL1 depletion led to heightened RAD18 expression, promoting chemoresistance possibly via enhancing Translesion DNA Synthesis (TLS)-mediated DNA lesion bypass. Moreover, dowregulation of NEIL1 results in reduced DNA damage accumulation and suppressed apoptosis in ovarian cancer. Overall, our findings unveil a novel mechanism involving NEIL1 and RAD18 in regulating chemoresistance in ovarian CSCs. Targeting this NEIL1-RAD18 axis may offer promising therapeutic strategies for combating chemoresistance and improving ovarian cancer treatment outcomes.

KLF8 is activated by TGF‐β1 via Smad2 and contributes to ovarian cancer progression

AbstractKrüppel‐like factor 8 (KLF8) is a transcription factor expressed abnormally in various cancer types and promotes oncogenic transformation. However, the role of KLF8 in ovarian cancer (OC) progression remains unclear. This study reports that transforming growth factor‐β1 (TGF‐β1)/Smad2/KLF8 axis regulates epithelial–mesenchymal transition (EMT) and contributes to OC progression. We analyzed the KLF8 expression in OC cells and tissues, wherein a significant overexpression of KLF8 was observed. Increased KLF8 expressions were correlated with higher cell proliferation, EMT, migration, and invasion and conferred poor clinical outcomes in OC patients. Overexpressed KLF8 increases F‐actin polymerization and induces cytoskeleton remodeling of OC cells. Furthermore, a dissection of the molecular mechanism defined that TGF‐β1 triggers KLF8 through the Smad2 pathway and regulates EMT. Pharmacological and genetic inhibition of Smad2 followed by TGF‐β1 treatment failed to activate KLF8 expression and induction of EMT. Using promoter‐luciferase reporter assays, we defined that upon TGF‐β1 activation, phosphorylated Smad2 binds and promotes the KLF8 promoter activity, and knockdown of Smad2 inhibits KLF8 promoter activation. Together, these results demonstrate that TGF‐β1 activates KLF8 expression by the Smad2 pathway, and KLF8 contributes to OC progression and may serve as a potential therapeutic strategy for treating OC patients.

MicroRNA-379-5p attenuates cancer stem cells and reduces cisplatin resistance in ovarian cancer by regulating RAD18/Polη axis

Abstract Ovarian cancer (OC) is an aggressive malignancy of the female reproductive organs, associated with a low 5-year survival rate. Emerging evidence suggests the pivotal role of microRNAs (miRNAs) in regulating chemoresistance and metastasis in OC, primarily through cancer stem cells (CSCs), also known as cancer stem-like cells (CSLCs). Herein, we demonstrate that miR-379-5p is downregulated in several OC cell populations including both cell lines and patient tumor samples. Furthermore, overexpression of miR-379-5p effectively inhibits CSCs and counteracts cisplatin-induced expansion of CSCs. Further mechanistic investigations identify RAD18, a DNA repair protein involved in translesion DNA synthesis (TLS), as a direct target of miR-379-5p. Moreover, a negative correlation between miR-379-5p and RAD18 expression is observed in ovarian CSCs isolated from OC patients. The downregulation of RAD18 inhibits stem-like phenotypes and enhances the sensitivity of ovarian CSCs to cisplatin treatment. Importantly, miR-379-5p-mediated inhibition of RAD18 prevents the repair synthesis in CSCs by promoting the accumulation of DNA damage. In vivo studies further reveal that miR-379-5p enhances DNA damage, which, in turn, inhibits tumor cell proliferation in athymic nude mice. Remarkably, targeting of RAD18 by miR-379-5p prevents monoubiquitination of proliferating cell nuclear antigen (PCNA), resulting in reduced DNA Polymerase η (a TLS polymerase that helps to bypass DNA lesions) recruitment to lesion sites. In the absence of Polη, the persisting DNA lesions cause activation of cell cycle arrest and apoptosis pathway in CSCs. Therefore, our findings unveil a novel mechanism whereby miR-379-5p overexpression curtails CSCs by modulating the RAD18/Polη axis.