Nanoenabled Intracellular Calcium Bursting for Safe and Efficient Reversal of Drug Resistance in Tumor Cells.

Multidrug resistance (MDR) of a tumor is the main cause of failure of clinical chemotherapy. Herein, we report a simple, yet versatile, tumor-targeting "calcium ion nanogenerator" (TCaNG) to reverse drug resistance by inducing intracellular Ca2+ bursting. Consequently, the TCaNG could induce Ca2+ bursting in acidic lysosomes of tumor cells and then reverse drug resistance according to the following mechanisms: (i) Ca2+ specifically accumulates in mitochondria, suppressing cellular respiration and relieving tumor hypoxia, thus inhibiting P-glycoprotein biosynthesis by downregulating HIF-1α expression. (ii) Ca2+-bursting-induced respiratory depression blocks intracellular ATP production, which further leads to the P-gp incompetence. As a result, the TCaNG could decrease the IC50 of DOX to MCF-7/ADR cells by approximately 30 times and reduce the proliferation of drug-resistant tumors by approximately 13 times without obvious side effects. This simple, safe, and effective "Ca2+ bursting" strategy holds the potential for clinical application in tumor treatment.

Inhibition of IGF1R signaling abrogates resistance to afatinib (BIBW2992) in EGFR T790M mutant lung cancer cells.

Non-small cell lung cancer (NSCLC) patients with an epidermal growth factor receptor (EGFR) mutation have benefited from treatment of reversible EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Acquisition of a secondary mutation in EGFR T790M is the most common mechanism of resistance to first generation EGFR TKIs, resulting in therapeutic failure. Afatinib is a second generation of EGFR TKI that showed great efficacy against tumors bearing the EGFR T790M mutation, but it failed to show the improvement on overall survival of lung cancer patients with EGFR mutations possibly because of novel acquired resistance mechanisms. Currently, there are no therapeutic options available for lung cancer patients who develop acquired resistance to afatinib. To identify novel resistance mechanism(s) to afatinib, we developed afatinib resistant cell lines from a parental human-derived NSCLC cell line, H1975, harboring both EGFR L858R and T790M mutations. We found that activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway contributes to afatinib resistance in NSCLC cells harboring the T790M mutation. IGF1R knockdown not only significantly sensitizes resistant cells to afatinib, but also induces apoptosis in afatinib resistance cells. In addition, combination treatment with afatinib and linsitinib shows more than additive effects on tumor growth in in vivo H1975 xenograft. Therefore, these finding suggest that IGF1R inhibition or combination of EGFR-IGF1R inhibition strategies would be potential ways to prevent or potentiate the effects of current therapeutic options to lung cancer patients demonstrating resistance to either first or second generation EGFR TKIs.

Synergistic Anti-Tumor Efficacy Achieved by Reversing Drug Resistance through the Regulation of the Tumor Immune Microenvironment with IL-12 and Osimertinib Combination Therapy.

The objective of this study was to investigate the role of IL-12 in enhancing the anti-tumor efficacy of the small molecule targeted drug osimertinib in resistant tumor models and reversing resistance mechanisms. We utilized paired non-small cell lung cancer H1975 tumor tissues, establishing mouse tumor models with diverse tumor immune microenvironments. Analytical methods including immunohistochemistry and immunofluorescence were employed to compare immune cell infiltration, cytokines, effector molecules, and protein changes in resistant signaling pathways in tumor tissues, shedding light on IL-12's mechanism of action in enhancing osimertinib efficacy and reversing resistance. Results showed that osimertinib monotherapy had limited tumor suppression, whereas IL-12 exhibited more significant anti-tumor effects. Combination therapy groups demonstrated even greater tumor suppression with increased immune cell infiltration, elevated immune-related factor secretion, reduced immunosuppressive MDSCs, and decreased resistance-related signaling pathway markers. In conclusion, IL-12 enhances anti-tumor efficacy and reverses osimertinib resistance through various mechanisms, including increased immune cell infiltration, reduced immunosuppressive MDSCs, enhanced immune cell granzyme and IFN-γ release, decreased PDL-1 expression, improved tumor microenvironment, restored immune surveillance, and heightened cancer cell sensitivity to osimertinib.

Targeting cancer drug resistance utilizing organoid technology.

Cancer organoids generated from 3D in vitro cell cultures have contributed to the study of drug resistance. Maintenance of genomic and transcriptomic similarity between organoids and parental cancer allows organoids to have the ability of accurate prediction in drug resistance testing. Protocols of establishing therapy-sensitive and therapy-resistant organoids are concluded in two aspects, which are generated directly from respective patients' cancer and by induction of anti-cancer drug. Genomic and transcriptomic analyses and gene editing have been applied to organoid studies to identify key targets in drug resistance and FGFR3, KHDRBS3, lnc-RP11-536 K7.3 and FBN1 were found to be key targets. Furthermore, mechanisms contributing to resistance have been identified, including metabolic adaptation, activation of DNA damage response, defects in apoptosis, reduced cellular senescence, cellular plasticity, subpopulation interactions and gene fusions. Additionally, cancer stem cells (CSCs) have been verified to be involved in drug resistance utilizing organoid technology. Reversal of drug resistance can be achieved by targeting key genes and CSCs in cancer organoids. In this review, we summarize applications of organoids to cancer drug resistance research, indicating prospects and limitations.

Cell- and subcellular organelle-targeting nanoparticle-mediated breast cancer therapy.

Breast cancer (BC) is the most prevalent malignant tumor, surpassing lung cancer as the most frequent malignancy in women. Drug resistance, metastasis, and immune escape are the major factors affecting patient survival and represent a huge challenge in BC treatment in clinic. The cell- and subcellular organelle-targeting nanoparticles-mediated targeted BC therapy may be an effective modality for immune evasion, metastasis, and drug resistance. Nanocarriers, efficiently delivering small molecules and macromolecules, are used to target subcellular apparatuses with excellent targeting, controlled delivery, and fewer side effects. This study summarizes and critically analyzes the latest organic nanoparticle-mediated subcellular targeted therapeutic based on chemotherapy, gene therapy, immunotherapy, and combination therapy in detail, and discusses the challenges and opportunities of nanoparticle therapy.

Design and synthesis of α-naphthoflavone chimera derivatives able to eliminate cytochrome P450 (CYP)1B1-mediated drug resistance via targeted CYP1B1 degradation.

Extrahepatic cytochrome P450 1B1 (CYP1B1), which is highly expressed in various tumors, is an attractive and potential target for cancer prevention, therapy, and reversal of drug resistance. CYP1B1 inhibition is the current predominant therapeutic paradigm to treating CYP1B1-mediated malignancy, but therapeutic effect has little success. Herein, we reported CYP1B1 degradation in place of CYP1B1 inhibition for reversing drug resistance toward docetaxel in CYP1B1-overexpressing prostate cancer cell line DU145 using a PROTAC strategy. Replacing chlorine atom of a CYP1B1 selective inhibitor we found previously with ethynyl, we got the resulting α-naphthoflavone derivative 5 which kept strong inhibition against CYP1B1 (IC50 = 0.4 ± 0.2 nM) and high selectivity. Coupling of 5 with thalidomide derivatives of varying chain lengths afforded conjugates 6A-Dvia click reaction. In vitro cell-based assay indicated that 6C was more effective in eliminating drug resistance of CYP1B1-overexpressed DU145 cells compared with other analogues. Western blotting analysis showed CYP1B1 degradation was one main reason for the reversal of drug resistance to docetaxel and the effect was obtained in a concentration-dependent manner. This work is the first attempt to overcome CYP1B1-mediated drug resistance via CYP1B1 degradation instead of CYP1B1 inhibition, which could provide a new direction toward eliminating drug resistance.

Choline Metabolism Alteration: A Focus on Ovarian Cancer.

Compared with normal differentiated cells, cancer cells require a metabolic reprograming to support their high proliferation rates and survival. Aberrant choline metabolism is a fairly new metabolic hallmark reflecting the complex reciprocal interactions between oncogenic signaling and cellular metabolism. Alterations of the involved metabolic network may be sustained by changes in activity of several choline transporters as well as of enzymes such as choline kinase-alpha (ChoK-α) and phosphatidylcholine-specific phospholipases C and D. Of note, the net outcome of these enzymatic alterations is an increase of phosphocholine and total choline-containing compounds, a "cholinic phenotype" that can be monitored in cancer by magnetic resonance spectroscopy. This review will highlight the molecular basis for targeting this pathway in epithelial ovarian cancer (EOC), a highly heterogeneous and lethal malignancy characterized by late diagnosis, frequent relapse, and development of chemoresistance. Modulation of ChoK-α expression impairs only EOC but not normal ovarian cells, thus supporting the hypothesis that "cholinic phenotype" is a peculiar feature of transformed cells and indicating ChoK-α targeting as a novel approach to improve efficacy of standard EOC chemotherapeutic treatments.

Global metabolic profile identifies choline kinase alpha as a key regulator of glutathione-dependent antioxidant cell defense in ovarian carcinoma.

Epithelial Ovarian Cancer (EOC) "cholinic phenotype", characterized by increased intracellular phosphocholine content sustained by over-expression/activity of choline kinase-alpha (ChoKα/CHKA), is a metabolic cellular reprogramming involved in chemoresistance with still unknown mechanisms.By stable CHKA silencing and global metabolic profiling here we demonstrate that CHKA knockdown hampers growth capability of EOC cell lines both in vitro and in xenotransplant in vivo models. It also affected antioxidant cellular defenses, decreasing glutathione and cysteine content while increasing intracellular levels of reactive oxygen species, overall sensitizing EOC cells to current chemotherapeutic regimens. Natural recovering of ChoKα expression after its transient silencing rescued the wild-type phenotype, restoring intracellular glutathione content and drug resistance. Rescue and phenocopy of siCHKA-related effects were also obtained by artificial modulation of glutathione levels. The direct relationship among CHKA expression, glutathione intracellular content and drug sensitivity was overall demonstrated in six different EOC cell lines but notably, siCHKA did not affect growth capability, glutathione metabolism and/or drug sensitivity of non-tumoral immortalized ovarian cells. The "cholinic phenotype", by recapitulating EOC addiction to glutathione content for the maintenance of the antioxidant defense, can be therefore considered a unique feature of cancer cells and a suitable target to improve chemotherapeutics efficacy.

Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin.

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.