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Chemotherapy-induced neurotoxicity is a common adverse effect of cancer treatment. No medication has been shown to be effective in the prevention or treatment of chemotherapy-induced neurotoxicity. Using minoxidil as an initial template for structural modifications in conjunction with an in vitro neurite outgrowth assay, an image-based high-content screening platform, and mouse behavior models, an effective neuroprotective agent CN016 was discovered. Our results showed that CN016 could inhibit paclitaxel-induced inflammatory responses and infiltration of immune cells into sensory neurons significantly. Thus, the suppression of proinflammatory factors elucidates, in part, the mechanism of action of CN016 on alleviating paclitaxel-induced peripheral neuropathy. Based on excellent efficacy in improving behavioral functions, high safety profiles (MTD500 mg/kg), and a large therapeutic window (MTD/MED50) in mice, CN016 might have great potential to become a peripherally neuroprotective agent to prevent neurotoxicity caused by chemotherapeutics as typified by paclitaxel.

The therapeutic potential of 5-HD in mouse model

The image-based high-content screening platform

Chemotherapy-induced neurotoxicity is a common adverse effect of cancer treatment. No medication has been shown to be effective in the prevention or treatment of chemotherapy-induced neurotoxicity. This study aimed to discover potential neuroprotective drugs for paclitaxel-induced neurotoxicity. An image-based high-content platform was first developed to screen for potential neuroprotective drugs. The screening system comprised of automated image acquisition and multiparameter analysis, including neuronal viability, neurite outgrowth, and synaptogenesis. By this platform, we obtained a candidate list from compound libraries. In the drug screening from compound libraries of ion channel ligands, REDOX and GABAergic ligands, 5-hydroxydecanoate (5-HD) exhibited the most significant neuroprotective effects against paclitaxel-induced neurotoxicity in both cortical and dorsal root ganglion (DRG) neurons. In mouse behavioral tests, 5-HD restored the thermal sensitivity and alleviated mechanical allodynia induced by paclitaxel. Electron micrographs of sciatic nerve revealed that 5-HD reduced the damages caused by paclitaxel in the nonmyelinated and smaller myelinated fibers. The mechanistic study on DRG neurons suggested that 5-HD rescued the dysregulation of intracellular calcium homeostasis provoked by paclitaxel. Importantly, 5-HD did not jeopardize the antitumor effect of paclitaxel in tumor xenograft models. In conclusion, we established an imaged-based high-content screening platform and a protocol for verifying the neuroprotective effect in vivo, by which 5-HD was identified and validated as a potential neuroprotective drug for paclitaxel-induced neuropathy. Mol Cancer Ther; 14(10); 2206–14. ©2015 AACR.

Antibody information

Information of drug library

Flow chart for drug discovery

PDF - 361KB, Supplementary Materials and Methods; Table S1: Primary Antibodies and secondary antibodies; Figure S1: Effect of MPT0B098 on the organizations of cellular microtubule network; Figure S2: Effect of MPT0B098 on HIF-regulated genes in A549 cells; Figure S3: MPT0B098 inhibits HuR translocation from the nucleus to the cytoplasm in HONE-1 and PC3 cells.

Neuroprotective drugs screened from the image-based high-content platform

The neuroprotective effects of 5-HD in mouse model

Microtubule inhibitors have been shown to inhibit hypoxia-inducible factor-1α (HIF-1α) expression through inhibition translation or enhancing protein degradation. Little is known of the effect of microtubule inhibitors on the stability of HIF-1α mRNA. We recently discovered a novel indoline–sulfonamide compound, 7-aryl-indoline-1-benzene-sulfonamide (MPT0B098), as a potent microtubule inhibitor through binding to the colchicine-binding site of tubulin. MPT0B098 is active against the growth of various human cancer cells, including chemoresistant cells with IC50 values ranging from 70 to 150 nmol/L. However, normal cells, such as human umbilical vein endothelial cells (HUVEC), exhibit less susceptibility to the inhibitory effect of MPT0B098 with IC50 of 510 nmol/L. Similar to typical microtubule inhibitors, MPT0B098 arrests cells in the G2–M phase and subsequently induces cell apoptosis. In addition, MPT0B098 effectively suppresses VEGF-induced cell migration and capillary-like tube formation of HUVECs. Distinguished from other microtubule inhibitors, MPT0B098 not only inhibited the expression levels of HIF-1α protein but also destabilized HIF-1α mRNA. The mechanism of causing unstable of HIF-1α mRNA by MPT0B098 is through decreasing RNA-binding protein, HuR, translocation from the nucleus to the cytoplasm. Notably, MPT0B098 effectively suppresses tumor growth and microvessel density of tumor specimens in vivo. Taken together, our results provide a novel mechanism of inhibiting HIF-1α of a microtubule inhibitor MPT0B098. MPT0B098 is a promising anticancer drug candidate with potential for the treatment of human malignancies. Mol Cancer Ther; 12(7); 1202–12. ©2013 AACR.

PDF file - 515KB, Body weight change of the mice model. The body weight was recorded before treatment for the baseline, and throughout the period of experiment. The plots represent the curve of (A) the dose-dependent BGT226 study, and (B) the comparison study of individual PI3K and mTOR inhibitors.

Loss-of-DUSP2 results in the expansion of CSC-like population.

Table S1. CSC frequency measured by ELDA in vitro. Table S2. Multiple corrections for correlation p value. Table S3. Tumor incidence in limiting dilution assay (I). Table S4. Tumor incidence in limiting dilution assay (II). Table S5. Top 10 list of candidate drugs exert similar actions like DUSP2. Table S6: IC50 of B369, B390, and SAHA determined by different methods.

PDF file - 757KB, Effect of the cell growth and the intracellular signals by BGT226. (A) Growth inhibitory effect of BGT226, rapamycin and LY294002. FaDu and OECM1 cells were treated with each compound in the identical concentration-dependent manner. The result was plotted (X axis, logarithm scale of concentration; y axis, ratio). (B) Effect of BGT226 and the other PI3K/mTOR targeting agents in OECM1. In the left panel, cells were treated with BGT226 120 nM or LY294002 2μM in a time-dependent manner. In the right panel, cells were treated with various PI3K/mTOR targeting agents for 30 min before whole cell lysate collection (G, BGT226 120 nM; K, BKM120 2μM; Rad, RAD001, 0.5 or 2 nM; L, LY294002 2μM; Rapa, rapamycin 100 or 200 nM; C, control).

HDACi abolished hypoxia-induced cancer malignancy

Knockdown of DUSP2 facilitates xenograft tumor growth

Legends for supplementary figures S1-S7

Figure S3: PGE2 mediates loss-of-DUSP2-induced cancer stemness. Figure S4: Hypoxia-induced COX-2 expression is mediated by downregulation of DUSP2.

Supplementary Table 1. Antibodies and Primer list Supplementary Table 2. IPA results for HNSCC upstream transcriptional regulators Supplementary Table 3. Clinical characteristics of RUNX2 in validation HNSCC cohort Supplementary Table 4. Lung colony formation abilities among HNSCC cells Supplementary Table 5. Relative expression and hazard ratio of RUNX2 target genes in GSE37991 HNSCC cohort and TCGA HNSCC cohort Supplementary Table 6. Clinical characteristics of RUNX2 and INHBA expression among TCGA HNSCC cohort Supplementary Table 7. Prediction binding affinity between 14q32.2 miRNAs and RUNX2 3'-UTR in HNSCC Supplement Table 8. Transcription factor 3'UTR miR-376c MRE analysis and binding prediction analysis of INHBA promoter. Supplementary Table 9. IPA Interaction analysis between RUNX2 and TFs and revealed differential expressed genes in GSE37991 HNSCC patients

CSF-1-induced phosphorylation and endocytosis of CSF-1R in purified macrophages with different CSF1R c.1085 genotypes.

Cancer stem-like cells (CSC) evolve to overcome the pressures of reduced oxygen, nutrients or chemically induced cell death, but the mechanisms driving this evolution are incompletely understood. Here, we report that hypoxia-mediated downregulation of the dual specificity phosphatase 2 (DUSP2) is critical for the accumulation of CSC in colorectal cancer. Reduced expression of DUSP2 led to overproduction of COX-2–derived prostaglandin E2, which promoted cancer stemness via the EP2/EP4 signaling pathways. Genetic and pharmacological inhibition of PGE2 biosynthesis or signal transduction ameliorated loss-of-DUSP2–induced tumor growth and cancer stemness. Genome-wide profile analysis revealed that genes regulated by DUSP2 were similar to those controlled by histone deacetylase. Indeed, treatment with novel histone deacetylase inhibitors abolished hypoxia-induced DUSP2 downregulation, COX-2 overexpression, cancer stemness, tumor growth, and drug resistance. Our findings illuminate mechanisms of cancer stemness and suggest new cancer therapy regimens. Cancer Res; 77(16); 4305–16. ©2017 AACR.

Purpose: Targeting tumor-associated macrophages with colony-stimulating factor 1 receptor (CSF-1R) inhibition reveals a strategy for cancer therapy. Here, we studied the impact of CSF1R germline genetic variant on CSF-1R signaling and the susceptibility to CSF-1R inhibitors.Experimental designs: CSF1R germline genetic variants were studied in 140 cancer patients. CSF-1R phosphorylation, endocytosis, and macrophage polarization were measured as the response to CSF-1 stimulation. Tumor-associated macrophages in surgical specimens and sensitivity to CSF-1R inhibitors were used to determine macrophage function.Results: A CSF1R c.1085A>G genetic variant causing the change of histidine to arginine in the domain of receptor dimerization was identified as a high allele frequency in Eastern Asian population. Cancer patients with this variant allele had less M2-like tumor-associated macrophages accompanied by low VEGF expression in tumor tissues. Importantly, CSF1R genetic variant was significantly associated with disease-free survival in colorectal, endometrial, and ovarian cancer. In terms of differentiation, macrophages with CSF1R c.1085A>G genetic variant displayed a refractory response to CSF-1 stimulation and macrophage survival was sensitive to CSF-1R inhibitors with IC50 of 0.1 to 1 nmol/L range. On contrast, CSF-1 induced a prominent phosphorylation and rapid endocytosis of CSF-1R, leading to an M2-like dominant polarization in macrophages with CSF1R c.1085 genotype A_A, in which CSF-1R inhibitors of PLX3397, BLZ945, and GW2580 inhibited macrophage survival with IC50 of 10 to 100 nmol/L range.Conclusions: The CSF1R c.1085A>G genetic variant regulates tumor immunity by altering the polarization and function of macrophages. This genetic variant confers the sensitivity to CSF-1R inhibitors, implying as a biomarker in targeting CSF-1R signaling for cancer treatment. Clin Cancer Res; 23(20); 6021–30. ©2017 AACR.

CSF-1R endocytosis in macrophage with CSF1R c.1085A_G.

HDACi inhibits tumor growth and drug resistance

Knockdown of DUSP2 enhanced EMT process and migration/invasion ability

PDF file - 468KB, (A) The inhibitory effect of BGT226 in cetuximab-resistant Hep3B cell line. Growth inhibition assay was used in this study. After subculture, cells were treated with cetuximab or BGT226 at least three generation time. The results of cetuximab were represented as curve plot in the left panel, and the IC50 with BGT226 were shown in the right table. (B) Basal expression of PTEN in all tested cell lines. All cell lysate was extracted at the time in steady growth. (C) Combination treatment of BGT226 with radiation in FaDu cells. Clonogenic assay was applied for the cell survival study, with 400 cells implanted into each of 6-well plate. After attachment, BGT226 was administrated 2 h prior to the radiation, and then replaced with drug-free medium after 24 h. The result was then assessed after incubation for 10 days.

Epigenetic correlates of the head and neck cancer may illuminate its pathogenic roots. Through a gene set enrichment analysis, we found that the oncogenic transcription factor RUNX2 is widely upregulated in the head and neck squamous cell carcinoma (HNSCC) with lymph node metastasis, where it also predicts poor prognosis in patients with HNSCC. Enforced expression of ectopic RUNX2 promoted the metastatic capabilities of HNSCC, whereas RUNX2 silencing inhibited these features. Mechanistic investigations showed that manipulating levels of activin A (INHBA) could rescue or compromise the RUNX2-mediated metastatic capabilities of HNSCC cells. Furthermore, we found that miR-376c-3p encoded within the 3′-untranslated region of RUNX2 played a pivotal role in regulating RUNX2 expression in highly metastatic HNSCC cells, where it was downregulated commonly. Restoring miR-376c expression in this setting suppressed expression of RUNX2/INHBA axis along with metastatic capability. Clinically, we observed an inverse relationship between miR-376c-3p expression and the RUNX2/INHBA axis in HNSCC specimens. In summary, our results defined a novel pathway in which dysregulation of the RUNX2/INHBA axis due to miR-376c downregulation fosters lymph node metastasis in HNSCC. Cancer Res; 76(24); 7140–50. ©2016 AACR.

CSF-1R endocytosis in macrophage with CSF1R c.1085 A_A.

iNOS expression in macrophages with CSF1R c.1085 genotype A_A and A_G.

Clinical characteristics of colorectal cancer patients.

PDF file - 1659KB, Apoptosis and autophagy-associated reaction by BGT226. (A) Apoptotic effect of BGT226. HSC3 cells incubated with either BGT226 or cisplatin for 24 h were analyzed for apoptosis-associated protein expression. Arrows indicated the cleavage-form of protein. (B) Effect of AVOs formation in FaDu cells treated with BGT226. Cells were stained with acridine orange. The extent of staining intensity in tested cells was analyzed and plotted by flow cytometry. The left rows represented cells incubated in DMSO or 3MA. The right row showed cells that were treated with BGT226 120 nM with or without the co-treatment of 3MA. The percentage of detected cells in each quadrant is marked on the panel. (C) Evaluation of cathepsin B/D/L (Cat B/D/L) activity in FaDu cells treated with BGT226. The activity of each factor was assessed by its individual activity kit (BioVisiion, CA). For the preparation, we followed manifacturer's protocol after BGT226 treatment of the cells. The samples were then transferred to 96-well plate, with the result read using fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. The data was then compared to the control, with the ratio ploted and shown in the bar chart.

Supplementary Figure 1. RUNX2 is nucleus location protein among HNSCC cells. Supplementary Figure 2. RUNX2 was a poor prognostic marker in HNSCC. Supplementary Figure 3. Lung colony formation ability of HNSCC cells. Supplementary Figure 4. RUNX2 and its regulation targets, INHBA, promoted HNSCC invasion and migration assay in HNSCC cells. Supplementary Figure 5. The TCGA-HNSCC dataset shows the positive correlation between RUNX2 and INHBA and their prognostic value. Supplementary Figure 6. Ectopic anti-miR-376c-3p increases RUNX2 3'-UTR reporter assay in Ca9-22 cells. Supplementary Figure 7Knockdown RUNX2 prevent anti-miR-376c-3p induced cell migration and invasion ablilties in Ca9-22 cells.

CSF1R c.1085A>G genetic variant in patients with different cancer types and different populations.

The clinical characteristics and survival difference of endometrial and ovarian cancer patients with CSF1R c.1085 genotype A_A and A_G.

The distribution of M1 and M2 macrophages in tumor specimens of endometrial and ovarian cancer.

PDF file - 652K, Entinostat or sodium butyrate does not affect SOCE activation and STIM1 trafficking.

Supplementary Information. Include supplementary material, methods, table title and figure legends

PDF file - 836K, The integrity of microtubules is necessary for STIM1 membrane trafficking and the interaction with Orai1.

PDF file - 732K, HDAC6 and a-tubulin deacetylation in cervical cancer cells.

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PDF file - 155K, Additional experimental procedures including RNA interference, Total internal reflection fluorescence (TIRF) microscopy, and cell proliferation and migration assay. Also includes description of antibodies and reagents used.

PDF file - 738K, HDAC6 inhibitor, tubastatin-A, blocks STIM1 membrane trafficking.

PDF file - 1456K, HDAC6 is involved in STIM1 trafficking, proliferation and migration of cervical cancer cells.

AVI file - 2482K

AVI file - 3670K

Supplementary Tables S1-S4. Supplementary Table S1: Clinicopathological characteristics of 40 pairs of OSCC specimens and their corresponding non-tumorous epithelia. Supplementary Table S2: A list of deregulated miRNA in 40 pairs of OSCC specimens and their corresponding non-tumorous epithelia. Supplementary Table S3: Characteristics of 83 OSCC patients for Wnt-7b IHC study. Supplementary Table S4: Top ten of enriched transcriptional factors in miRNA overexpression.

Supplementary Figures S1-S7. Supplementary Figure S1: Genome-wide profiling of chromosome 14q32.2 miRNA on OSCC tissues. Supplementary Figure S2: Mi-RNA level in miR-PM overexpressed OEC-M1 and SCC-15 cells. Supplementary Figure S3: Effect of miR-329 and miR-410 on Wnt/β-catenin signaling pathway. Supplementary Figure S4: The effects of miR-329 and miR-410 on tumor cell proliferation. Supplementary Figure S5: Expression level of miRNA in xenograft tumor tissue. Supplementary Figure S6: Effects of miR-329 and -410 on tumorigenicity. Supplementary Figure S7: Effects of arecoline on Wnt/β-catenin signaling pathway.

Salvinal is a natural lignan isolated from the roots of Salvia mitorrhiza Bunge (Danshen). Previous studies have demonstrated its anti-proliferative activity in both drug-sensitive and -resistant cancer cell lines, with IC50 values ranging from 4–17 µM. In this study, a series of salvinal derivatives was synthesized and evaluated for the structure–activity relationship. Among the twenty-four salvinal derivatives, six compounds showed better anticancer activity than salvinal. Compound 25 displayed excellent anticancer activity, with IC50 values of 0.13–0.14 µM against KB, KB-Vin10 (overexpress MDR/Pgp), and KB-7D (overexpress MRP) human carcinoma cell lines. Based on our in vitro microtubule depolymerization assay, compound 25 showed depolymerization activity in a dose-dependent manner. Our findings indicate that compound 25 is a promising anticancer agent with depolymerization activity that has potential for the management of malignance.

Background Lymph node and distant metastasis contribute to poor outcomes in patients with oral squamous cell carcinoma (OSCC). The mechanisms regulating cancer migration and invasion play a key role in OSCC. Methods We determined migration and invasion ability of OSCC by wound-healing assay, two-chamber transwell invasion assay and cell mobility tracking and evaluated tumor metastasis in vivo. Western blot (WB), qRT-PCR, RNA-seq, dual-luciferase reporter assays and nuclear/cytoplasmic fractionation were performed to investigate the potential mechanism. Immunohistochimical (IHC) staining determined vimentin and PDZK1IP1 expression in OSCC tissues. Results and conclusion In this study, we determined that miR-455-5p was associated with lymph node metastasis and clinical invasion, leading to poor outcomes in patients with OSCC. MiR-455-5p promoted oral cancer cell migration and invasion and induced epithelial-to-mesenchymal transition (EMT). We also identified a new biomarker, PDZK1IP1 (MAP17), that was targeted by miR-455-5p. PDZK1IP1 knockdown led to migration, metastasis, EMT, and increased transforming growth factor-β signaling in OSCC. In addition, miR-455-5p overexpression and PDZK1IP1 inhibition promoted collective OSCC cell migration. According to data from the Cancer Genome Atlas database and the NCKU-OrCA-40TN data set, miR-455-5p and PDZK1IP1 are positively and negatively correlated, respectively, with partial EMT score. High miR-455-5p expression was associated with high vimentin levels and low MAP17 H-scores. The patients with low MAP17 expression had higher rates of disease recurrence than did patients with high MAP17 expression, especially for patients with clinical invasion risk factors and low MAP17 expression. These results suggest that miR-455-5p suppresses PDZK1IP1 expression and mediates OSCC progression. MiR-455-5p and PDZK1IP1 may therefore serve as key biomarkers and be involved in regulating partial EMT in OSCC cells. PDZK1IP1 expression may also serve as an independent factor that impacts outcomes in patients with clinical risk factors for recurrence.