Despite initial positive responses with chemotherapy, many cancer patients experience relapse, continued tumor growth, and metastatic spread due to drug resistance. It is well documented that a rare population of phenotypically heterogeneous cells contributes to intratumour heterogeneity and drug resistance. To date, these rare populations are poorly characterized. To identify the potential role of these rare populations in drug resistance, here we have performed single-cell RNA sequencing of human oral squamous cell carcinomas lines presenting with sensitive, early, and late cisplatin-resistance patterns. The single-cell RNA-sequencing data identified two different transitional clusters within the three, sensitive, early, and late cisplatin-resistant major clusters. The differential gene expression profile and deregulated pathways analysis suggested Brain Abundant Membrane-Attached Signal Protein 1 (BASP1) as a major upregulated gene not only in major drug-resistant clusters but also in transitional clusters. Selective knockdown of BASP1 reverses epithelial to mesenchymal transition (EMT) phenotype in cisplatin-resistant cells and restores cisplatin-induced cell death. Mechanistically, BASP1 positively regulates LIN7A expression through phosphorylation of RAC-alpha serine/threonine-protein kinase as well as by supressing microRNA hsa-mir-501-3p, which in turn induces β-catenin-mediated EMT in chemoresistant cells. Overall, our study demonstrates that BASP1 acts as a key regulator of EMT in cisplatin-resistant oral squamous cell carcinoma and represents a promising therapeutic target to overcome drug resistance in advanced stages of the disease.

Cisplatin and its analogues are valuable anti-cancer drugs that target the genome, block DNA replication, and induce apoptosis. As a counteractive response, cancer cells activate several mechanisms to maintain uninterrupted DNA replication, and those are yet to be fully elucidated. This study using head and neck squamous carcinoma cells (HNSCC) demonstrated the involvement of DNA polymerase Kappa (Polκ), a trans-lesion DNA synthesis (TLS) polymerase that primarily functions as a mismatch extender, in cisplatin resistance. Interestingly, the catalytic activity of Polκ plays a minimal role in adduct bypass; rather, tripartite interactions involving it, rewire and stabilize the stalled replication fork. While the Polκ-PCNA-Polδ axis facilitates efficient proliferation of cisplatin-resistant cells, the Polκ-PCNA-USP18 axis stabilizes critical proteins of ATM-ATR, and HR and NHEJ pathways to protect replication fork, repair damage, and restart DNA synthesis under cisplatin-induced stress. In resistant cells, the efficiency of ubiquitin-mediated proteasomal degradation is low, which is further diminished by Polκ-recruited USP18 deubiquitinase, maintaining a cellular homeostasis. In conclusion, for the first time, we uncovered two critical Polκ axes crucial for regulating cisplatin toxicity in cells and provided foundation for future drug discovery against advance HNSCC by targeting this non-essential DNA polymerase.

Chemoresistance poses a significant challenge while treating triple-negative breast cancer, and cancer stem cells are one of the key regulators of chemoresistance in breast cancer. SOX2, a crucial regulator of pluripotency in breast cancer stem cells, has been associated with poor prognosis and therapeutic resistance. Breast cancer stem cells can evade chemotherapy by modulating DNA damage pathways and altering cell cycle regulation. However, how SOX2 is linked with these pathways is largely unknown, which is the primary focus of our study. Overexpression clones were established for cell lines with low SOX2 expression to conduct gain-of-function experiments, while loss-of-function studies were performed on high SOX2-expressing lines using SOX2 silencer RNA (siRNA). SOX2 overexpression in breast cancer enhanced migration, sphere formation, and chemoresistance, while SOX2 knockdown inhibited these processes. Similar effects were observed on the expression of drug efflux proteins (ABCB1, ABCC1, ABCG2). Flow cytometry analysis revealed significant changes in the proportion of cells in the G2/M phase when SOX2 was overexpressed or knocked down, both in the presence and absence of doxorubicin, at varying doses. Immunoblotting confirmed that SOX2 overexpression induced a G2/M cell cycle arrest, as well as DNA damage response and repair. In nude mice, SOX-2 overexpression resulted in an enhanced capacity to form breast cancer tumors compared with the empty vector control. These findings illustrated that targeting SOX2 could be a promising strategy for overcoming chemoresistance in breast cancer. Overall, this study suggests that targeting SOX2 may be an effective strategy for overcoming chemoresistance in breast cancer.

BACKGROUND:Deregulated DNA damage response (DDR) network is implicated in cancer progression and therapy resistance. OBJECTIVE:The present study was designed to investigate whether nimbolide, an anticancer neem limonoid, targets key components of the DDR signalling pathway in cellular and animal models of oral squamous cell carcinoma (OSCC). METHODS:OSCC cells (SCC-4 and SCC-9), 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinoma model, chemoresistant OSCC patient-derived xenograft (PDX) model established in athymic nude mice, and tissue sections from patients with oral premalignant/malignant disease were used for the study. Key molecules that orchestrate the DDR, including the MRN complex, ATM, DNA-PKcs, H2AX, and p53, were analysed by qRTPCR, immunoblotting, immunofluorescence, and immunohistochemistry. Cell proliferation and apoptosis indices were evaluated. RESULTS:Nimbolide significantly reduced 8-oxodG levels, expression of MRN, ATMS1891, and γ- H2AX, with an increase in p-p53S15 in OSCC cells as well as in the HBP model. Nimbolide potentiated the effect of KU-55933 in ATM inhibition. In the PDX model, nimbolide suppressed tumor formation, stimulated DDR and apoptosis, inhibited cell proliferation, and enhanced sensitivity to cisplatin. Analysis of p-ATM expression revealed a significant increase during the sequential progression of hamster and human OSCC. CONCLUSION:This study provides compelling evidence that nimbolide functions as a DDR inhibitor in cellular and hamster OSCC models and as a DDR activator in the PDX model primarily by targeting ATM. Small molecules like nimbolide that modulate DDR are of immense benefit in cancer therapy. The study has also unveiled p-ATM as a promising biomarker of tumour progression in human OSCCs.

Docetaxel is the most common chemotherapy regimen for several neoplasms, including advanced OSCC (Oral Squamous Cell Carcinoma). Unfortunately, chemoresistance leads to relapse and adverse disease outcomes. We performed CRISPR-based kinome screening to identify potential players of Docetaxel resistance. Immunohistochemistry was performed to examine the expression profile of the target gene across tumour tissues. Global transcriptome analysis was performed to determine the molecular mechanism underlying Docetaxel resistance. NEK9 kinase assay was performed to identify a putative kinase inhibitor. Upon conducting CRISPR-based kinome screening, Never In Mitosis Gene-A Related Kinase-9 (NEK9) was identified as a major player of Docetaxel resistance in OSCC, prostate, and pancreatic cancer lines. NEK9 expression was found to be upregulated in chemotherapy non-responder OSCC patients as compared to responders. NEK9 ablation restores Docetaxel-induced cell death in chemoresistant cells. Mechanistically, we found that NEK9 deletion upregulates Transducin-like enhancer protein 3 (TLE3), which in turn represses Wnt signalling. Fostamatinib was identified as a potent NEK9 inhibitor that overcomes Docetaxel resistance. Our study demonstrated that NEK9 plays an important role in Docetaxel resistance. The novel combination of NEK9 inhibitor Fostamatinib and Docetaxel needs further clinical investigation in advanced OSCC.

Chemoresistance is one of the major factors for treatment failure in OSCC.Reprogramming chemoresistance cells to undergo drug induced apoptotic cell death is a feasible approach to overcome drug resistance.Cyanobacteria is considered important sources of lead compounds for the development of drugs for treating cancer chemoresistance.This study deals with the role of Tolypothrix Dichloromethane Ethyl acetate fraction (TDEF) inducing apoptosis in cisplatin resistance H357 cell (H357cisR) and the underlying mechanisms sensitizing the chemoresistance.TDEF showing effective activity against H357cisR with IC 50 -14.13±1.18µg mL -1 , inhibits proliferation and migration.Proteome apoptosis arrays were found to stimulate phosphorylation of p53, activation of proapoptotic proteins including BAX and cytochrome C (CYCS), caspase-3/9 (CASP3/9), suppression of anti-apoptotic proteins like Bcl2, survivin and increased expression of the cell cycle checkpoint protein p21, p27.TDEF induced apoptosis with cell death-transducing signals, that regulate the Matrix metalloproteinases (MMPs) by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol thus triggered the activation of caspases-9 to activate downstream executioner caspase-3/7 required for apoptotic changes.The mechanistic pathway of apoptotic cell death in H357cisR was done through inhibiting β-catenin through GSK3β in turn activated by AKT.The phosphorylated β-catenin leads to proteasome degradation and unable to translocation to nucleus thereby activating c-Myc, survivin, Cyclin D and upregulate p21 expression which lead to cell cycle arrest in G 0 /G 1 phase.

Docetaxel alone or in combination with other drugs is the most common chemotherapy regimen for several neoplasms including advanced OSCC. Unfortunately, chemoresistance leads to relapse and continued tumor growth. It is therefore important to explore the causative factors for docetaxel resistance. In this study, we performed a CRISPR-based kinome screening that identified Never In Mitosis Gene-A Related Kinase-9 (NEK9) as a major player of docetaxel resistance in OSCC, prostate, and pancreatic cancer lines. NEK9 expression was upregulated in tumor samples of chemotherapy non-responders compared to responder OSCC patients. Our validation data suggests selectively knocking out NEK9 sensitizes cancer cells to docetaxel. Mechanistically, we found that ablation of NEK9 induces DNA damage, activating ERK(p-T202/Y204) that leads to Gasdermin-E mediated Cancer Cell pyroptosis. The in-vitro kinase activity assay identified fostamatinib as a potent inhibitor of NEK9. The xenograft data suggest that fostamatinib restores docetaxel sensitivity and facilitates a significant reduction of tumor burden. Overall, our data suggests a novel combination of fostamatinib and docetaxel needs further clinical investigation in advanced OSCC. ### Competing Interest Statement The authors have declared no competing interest.

Cisplatin alone or in combination with 5FU and docetaxel is the preferred chemotherapy regimen for advancedstage OSCC patients. However, its use has been linked to recurrence and metastasis due to the development of drug resistance. Therefore, sensitization of cancer cells to conventional chemotherapeutics can be an effective strategy to overcome drug resistance. Piperlongumine (PL), an alkaloid, have shown anticancer properties and sensitizes numerous neoplasms, but its effect on OSCC has not been explored. However, low aqueous solubility and poor pharmacokinetics limit its clinical application. Therefore, to improve its therapeutic efficacy, we developed piperlongumine-loaded PLGA-based smart nanoparticles (smart PL-NPs) that can rapidly release PL in an acidic environment of cancer cells and provide optimum drug concentrations to overcome chemoresistance. Our results revealed that smart PL-NPs has high cellular uptake in acidic environment, facilitating the intracellular delivery of PL and sensitizing cancer cells to cisplatin, resulting in synergistic anticancer activity in vitro by increasing DNA damage, apoptosis, and inhibiting drug efflux. Further, we have mechanistically explored the Hippo-YAP signaling pathway, which is the critical mediator of chemoresistance, and investigated the chemosensitizing effect of PL in OSCC. We observed that PL alone and in combination with cisplatin significantly inhibits the activation of YAP and its downstream target genes and proteins. In addition, the combination of cisplatin with smart PL-NPs significantly inhibited tumor growth in two preclinical models (patient-derived cell based nude mice and zebrafish xenograft). Taken together, our findings suggest that smart PL-NPs with cisplatin will be a novel formulation to reverse cisplatin resistance in patients with advanced OSCC.

PDF file - 275K, Characterization of stable shmda-9/syntenin and mda-9/syntenin clones.

PDF file - 212K, Human angiogenesis array coordinates.

<p>Supplementary Figures 1-9. Sabutoclax has greater efficacy than ABT-737. (S1) Sabutoclax causes a G1-S phase cell cycle arrest. (S2) Pancreatic cancer cells exhibit varying levels of sensitivity to Minocycline. (S3) Sabutoclax and Minocycline induce cytotoxicity in PANC-1 cells that is reversed with zVAD. (S4) Sabutoclax and Minocycline show synergy. Combination index (CI) values for the combination of Sabutoclax and Minocycline in MIA PaCa-2 cells. (S5) The cytotoxicity induced by Sabutoclax and Minocycline is caspase-dependent and dependent upon loss of Stat3 activation. (S6) Sabutoclax and Minocycline reduce tumor growth in a subcutaneous xenograft model of pancreatic cancer. (S7) Tumors from Pdx-1-Cre/K-rasLSL-G12D/p53flox/wt and Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox mice overexpress Mcl-1 and show sensitivity to Sabutoclax and Minocycline. (S8) Tumors from KPC (Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox )mice treated with Sabutoclax and Minocycline show decreased Stat3 activation. (S9)</p>

PDF file - 255K, S2. siRNA-mediated silencing of IL-20R2 or IL-22R receptor pairs in DU-145 and HO-1 cells.

PDF file - 89K, Graphical presentation of CD-31 expression in tumor sections.

PDF file - 264K, Quantitation of protein expression.

PDF file - 5906K, Supplemental Table I - Inhibition of mammary carcinogenesis in Wistar-Furth rats by mda-7/IL-24 Supplemental Table II - Microarray analysis of differential gene expression in FE1.2 + IL-24S compared to FE1.2 + IL-24AS cells. Supplemental Figure 1: Serum starvation induces GAS3 expression in both FE1.2 and FE1.3 cells. Supplemental Figure 2: GAS3 downregulation decreases the growth of cells in soft agar. Supplemental Figure 3: STAT3 inhibition reduced attachment of attaches cells to culture plates. Supplemental Figure 4: GAS3 expression reduced attachment of confluent cells to culture plates. Supplemental Figure 5: Binding of GAS3 to β1 integrin using immunoprecipitation assay. Supplemental Figure 6: GAS3 expression inhibits attachment to and proliferation on fibronectin coated culture plates. Supplemental Figure 7: GAS3 expression increases attachment to laminin coated culture plates. Supplemental Figure 8: GAS3 expression by immunohistochemistry.

Supplementary Figure 1, Table 1 from Mechanism by Which Mcl-1 Regulates Cancer-Specific Apoptosis Triggered by mda-7/IL-24, an IL-10–Related Cytokine

PDF file - 746K, Quantitation of protein expression.

PDF file - 174K, S1. Expression of IL-20R1, IL-20R2 and IL-22R receptors in A549, DU-145 and HO-1 cells.