Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals.

While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.

Transcriptome-Wide Identification and Quantification of Caffeoylquinic Acid Biosynthesis Pathway and Prediction of Its Putative BAHDs Gene Complex in A. spathulifolius.

The phenylpropanoid pathway is a major secondary metabolite pathway that helps plants overcome biotic and abiotic stress and produces various byproducts that promote human health. Its byproduct caffeoylquinic acid is a soluble phenolic compound present in many angiosperms. Hydroxycinnamate-CoA shikimate/quinate transferase is a significant enzyme that plays a role in accumulating CQA biosynthesis. This study analyzed transcriptome-wide identification of the phenylpropanoid to caffeoylquinic acid biosynthesis candidate genes in A. spathulifolius flowers and leaves. Transcriptomic analyses of the flowers and leaves showed a differential expression of the PPP and CQA biosynthesis regulated unigenes. An analysis of PPP-captive unigenes revealed a major duplication in the following genes: PAL, 120 unigenes in leaves and 76 in flowers; C3'H, 169 unigenes in leaves and 140 in flowers; 4CL, 41 unigenes in leaves and 27 in flowers; and C4H, 12 unigenes in leaves and 4 in flowers. The phylogenetic analysis revealed 82 BAHDs superfamily members in leaves and 72 in flowers, among which five unigenes encode for HQT and three for HCT. The three HQT are common to both leaves and flowers, whereas the two HQT were specialized for leaves. The pattern of HQT synthesis was upregulated in flowers, whereas HCT was expressed strongly in the leaves of A. spathulifolius. Overall, 4CL, C4H, and HQT are expressed strongly in flowers and CAA and HCT show more expression in leaves. As a result, the quantification of HQT and HCT indicates that CQA biosynthesis is more abundant in the flowers and synthesis of caffeic acid in the leaves of A. spathulifolius.

Lineage-Specific Variation in IR Boundary Shift Events, Inversions, and Substitution Rates among Caprifoliaceae s.l. (Dipsacales) Plastomes.

Caprifoliaceae s.l. plastid genomes (plastomes) show that one inversion and two inverted repeat boundary shifts occurred in the common ancestor of this family, after which the plastomes are generally conserved. This study reports plastome sequences of five additional species, Fedia cornucopiae, Valeriana fauriei, and Valerianella locusta from the subfamily Valerianoideae, as well as Dipsacus japonicus and Scabiosa comosa from the subfamily Dipsacoideae. Combined with the published plastomes, these plastomes provide new insights into the structural evolution of plastomes within the family. Moreover, the three plastomes from the subfamily Valerianoideae exhibited accelerated nucleotide substitution rates, particularly at synonymous sites, across the family. The patterns of accD sequence divergence in the family are dynamic with structural changes, including interruption of the conserved domain and increases in nonsynonymous substitution rates. In particular, the Valeriana accD gene harbors a large insertion of amino acid repeat (AAR) motifs, and intraspecific polymorphism with a variable number of AARs in the Valeriana accD gene was detected. We found a correlation between intron losses and increased ratios of nonsynonymous to synonymous substitution rates in the clpP gene with intensified positive selection. In addition, two Dipsacoideae plastomes revealed the loss of the plastid-encoded rps15, and a potential functional gene transfer to the nucleus was confirmed.

Defective erythropoiesis caused by mutations of the thyroid hormone receptor α gene.

Patients with mutations of the THRA gene exhibit classical features of hypothyroidism, including erythroid disorders. We previously created a mutant mouse expressing a mutated TRα1 (denoted as PV; Thra1PV/+ mouse) that faithfully reproduces the classical hypothyroidism seen in patients. Using Thra1PV/+ mice, we explored how the TRα1PV mutant acted to cause abnormalities in erythropoiesis. Thra1PV/+ mice exhibited abnormal red blood cell indices similarly as reported for patients. The total bone marrow cells and erythrocytic progenitors were markedly reduced in the bone marrow of Thra1PV/+ mice. In vitro terminal differentiation assays showed a significant reduction of mature erythrocytes in Thra1PV/+ mice. In wild-type mice, the clonogenic potential of progenitors in the erythrocytic lineage was stimulated by thyroid hormone (T3), suggesting that T3 could directly accelerate the differentiation of progenitors to mature erythrocytes. Analysis of gene expression profiles showed that the key regulator of erythropoiesis, the Gata-1 gene, and its regulated genes, such as the Klf1, ß-globin, dematin genes, CAII, band3 and eALAS genes, involved in the maturation of erythrocytes, was decreased in the bone marrow cells of Thra1PV/+ mice. We further elucidated that the Gata-1 gene was a T3-directly regulated gene and that TRα1PV could impair erythropoiesis via repression of the Gata-1 gene and its regulated genes. These results provide new insights into how TRα1 mutants acted to cause erythroid abnormalities in patients with mutations of the THRA gene. Importantly, the Thra1PV/+ mouse could serve as a preclinical mouse model to identify novel molecular targets for treatment of erythroid disorders.

Inhibiting casein kinase 2 overcomes paclitaxel resistance in gastric cancer.

PURPOSE: Casein kinase (CK) 2 activation has been implicated in the proliferation of various tumor types and resistance to chemotherapy. We investigated the mechanistic basis for the association between CK2 activation and paclitaxel resistance in a gastric cancer (GC). EXPERIMENTAL DESIGN: CK2 expression was evaluated in 59 advanced GC patients treated with paclitaxel as the second-line therapy. The efficacy of a CK2 inhibitor, CX-4945, and paclitaxel was evaluated in GC cell lines and a xenograft model. RESULTS: Patients with high CK2 expression (29/59, 39%) showed lower disease control rates (47.7% vs. 72.3%, p = 0.017) and shorter progression-free survival (2.8 vs. 4.8 months, p = 0.009) than patients with low CK2 expression. CK2 protein expression was associated with sensitivity to paclitaxel in 49 GC cell lines. Combination therapy with CX-4945 and paclitaxel exerted synergistic antiproliferative effects and inhibited the downregulation of phosphatidylinositol 3-kinase/AKT signaling in SNU-1 cells. In the SNU-1 xenograft model, the combination treatment was significantly superior to either single agent, suppressing tumor growth without notable toxicities. CONCLUSIONS: These results demonstrated that CK2 activation was related to paclitaxel resistance and that CX-4945 in combination with paclitaxel could be used as a potential treatment for paclitaxel resistance in GC.

miR-200c regulates induction of apoptosis through CD95 by targeting FAP-1.

Tumor progression shares many characteristics with the process of epithelial-to-mesenchymal transition (EMT). Cells that have undergone an EMT are known to have an increased resistance to apoptosis. CD95/Fas is an apoptosis-inducing receptor expressed on many tissues and tumor cells. During tumor progression CD95 is frequently downregulated, and tumor cells lose apoptosis sensitivity. miR-200 microRNAs repress both the EMT-inducing ZEB1 and ZEB2 transcription factors. We now demonstrate that miR-200c sensitizes cells to apoptosis mediated by CD95. We have identified the apoptosis inhibitor FAP-1 as a target for miR-200c. FAP-1 was demonstrated to be responsible for the reduced sensitivity to CD95-mediated apoptosis in cells with inhibited miR-200. The identification of FAP-1 as an miR-200c target provides a molecular mechanism to explain both the downregulation of CD95 expression and the reduction in sensitivity of cells to CD95-mediated apoptosis that is observed in the context of reduced miR-200 expression during tumor progression.

Diffuse sclerosing variant of papillary thyroid carcinoma: major genetic alterations and prognostic implications.

AIM: Diffuse sclerosing variant of papillary thyroid carcinoma (DSV-PTC) is an uncommon variant of PTC, and its prognostic significance remains controversial. The aim of this study was to investigate the major genetic alterations of DSV-PTC and their prognostic implications. METHODS AND RESULTS: We included 37 patients with DSV-PTC who underwent thyroid surgery and had formalin-fixed paraffin-embedded samples. We tested for a panel of genetic alterations, including BRAF(V) (600E) , NRAS codon 61, HRAS codon 12/13/61 and KRAS codon 12/13 point mutations as well as RET/PTC1, RET/PTC3 and PAX8/PPARγ rearrangements using reverse transcription real-time polymerase chain reaction (PCR). All genetic alterations found on PCR were confirmed by Sanger sequencing. Associations between the identified genetic alterations and clinicopathological characteristics were evaluated. Among 37 cases of DSV-PTC, 17 were positive for RET/PTC1 (46%), six for RET/PTC3 (16%) and nine for BRAF(V) (600E) (24%). All mutations/rearrangements were mutually exclusive. The remaining five cases had none of the above genetic alterations. DSV-PTC with RET/PTC3 rearrangement was associated with advanced-stage disease, including T4 and distant metastasis (P < 0.05). Patients with RET/PTC3 showed a higher frequency of persistent disease (P < 0.01). In contrast, DSV-PTC with RET/PTC1 was associated with a higher prevalence of disease remission (P < 0.05) and coexistent Hashimoto's thyroiditis (P < 0.01). CONCLUSION: Taken together, RET/PTC rearrangement was the major genetic alteration seen in patients with DSV-PTC, and the RET/PTC3 rearrangement was associated with advanced stage at diagnosis and poor clinical outcome.

CD95 promotes tumour growth.

CD95 (also called Fas and APO-1) is a prototypical death receptor that regulates tissue homeostasis mainly in the immune system through the induction of apoptosis. During cancer progression CD95 is frequently downregulated or cells are rendered apoptosis resistant, raising the possibility that loss of CD95 is part of a mechanism for tumour evasion. However, complete loss of CD95 is rarely seen in human cancers and many cancer cells express large quantities of CD95 and are highly sensitive to CD95-mediated apoptosis in vitro. Furthermore, cancer patients frequently have elevated levels of the physiological ligand for CD95, CD95L. These data raise the possibility that CD95 could actually promote the growth of tumours through its non-apoptotic activities. Here we show that cancer cells in general, regardless of their CD95 apoptosis sensitivity, depend on constitutive activity of CD95, stimulated by cancer-produced CD95L, for optimal growth. Consistently, loss of CD95 in mouse models of ovarian cancer and liver cancer reduces cancer incidence as well as the size of the tumours. The tumorigenic activity of CD95 is mediated by a pathway involving JNK and Jun. These results demonstrate that CD95 has a growth-promoting role during tumorigenesis and indicate that efforts to inhibit its activity rather than to enhance it should be considered during cancer therapy.

VEGF-C mediates RhoGDI2-induced gastric cancer cell metastasis and cisplatin resistance.

Rho GDP dissociation inhibitor 2 (RhoGDI2) expression is correlated with tumor growth, metastasis and chemoresistance in gastric cancer. However, the mechanisms by which RhoGDI2 promotes tumor cell survival and metastasis remain unclear. In this study, we clearly demonstrate that RhoGDI2 upregulates VEGF-C expression and RhoGDI2 expression is positively correlated with VEGF-C expression in human gastric tumor tissues as well as parental gastric cancer cell lines. VEGF-C depletion suppressed RhoGDI2-induced gastric cancer metastasis and sensitized RhoGDI2-overexpressing cells to cisplatin-induced apoptosis in vitro and in vivo. Secreted VEGF-C enhanced gastric cancer cell invasion and conferred cisplatin resistance to RhoGDI2-overexpressing cells. We also show that RhoGDI2 positively regulates Rac1 activity in gastric cancer cells. Inhibition of Rac1 expression suppressed RhoGDI2-induced VEGF-C expression, and this inhibition was associated with decreased invasiveness and increased sensitivity to cisplatin in RhoGDI2-overexpressing cells. Our results indicate that RhoGDI2 might be a potential therapeutic target for simultaneously reducing metastasis risk and enhancing chemotherapy efficacy in gastric cancer.

Promoter mutation-independent TERT expression is related to immune-enriched milieu in papillary thyroid cancer.

Telomerase reverse transcriptase promoter mutation (pTERT MT) promotes human carcinogenesis via aberrant expression of telomerase reverse transcriptase (TERT). However, the tumorigenic impact of TERT expression independent of pTERT MT remains unclear despite numerous mechanisms of TERT being suggested. To tackle this issue, we employed comprehensive bioinformatics to assess biological variations noticed among different TERT expression mechanisms. Papillary thyroid cancer (PTC) with pTERT MT (pTERT MT PTC) presented aggressive clinical behavior and exhibited biological profiles associated with cellular immortality and genomic instability. PTC with TERT expression, but without pTERT MT (TERT (+) PTC), also exhibited poor clinicopathological characteristics and was enriched with immune responses. In accordance, c-MYC/E2F and nuclear factor kappa B (NFκB) were dominant transcription factors in pTERT MT PTC and TERT (+) PTC, respectively. Notably, we revealed TERT hypermethylated oncological region (THOR) as potential TERT expressing mechanism in TERT (+) PTC patients. Furthermore, three unique subtypes of papillary thyroid cancer were deciphered using combination of machine learning based scoring systems. Our proposed scoring system was clinically significant especially in microcarcinoma predicting survival outcomes and inferring therapeutic responses of radioactive iodine therapy. Finally, our analysis was expanded to endocrine-related cancers, unveiling various regulatory mechanisms of TERT with poor clinical outcomes and biological behaviors.

Expression of adiponectin receptor 1 is indicative of favorable prognosis in non-small cell lung carcinoma.

Lung cancer is a major cause of cancer-related death worldwide. It is believed that obesity-related malignancies such as breast, endometrial, colorectal, and kidney carcinomas have lower plasma level and/or tissue expression of adiponectin receptors. However, the association between adiponectin receptors and lung cancer, a non obesity-related malignancy, is still unknown. We evaluated the tissue expression of adiponectin receptor (AdipoR) 1 and AdipoR2 in 83 cases of non-small cell lung carcinoma (NSCLC) and matched non-neoplastic lung tissues by immunohistochemistry and real-time polymerase chain reaction (PCR). Clinicopathological data, including smoking history, smoker's bronchiolitis, emphysema, lymph node metastasis, and T-stage were collected and evaluated. Expression of immunohistochemically stained AdipoR1 and AdipoR2 was observed in all samples of non-neoplastic lung tissues. Both receptors showed higher mRNA expression in non-neoplastic than neoplastic tissues (p < 0.05). In NSCLC tissues, AdipoR1 immunohistochemical expression was not observed in most of patients with squamous cell carcinoma and current smoking history (31/42, p = 0.04 and 25/29, p = 0.003, respectively). Additionally, AdipoR1 mRNA expression was significantly lower in patients with lymph node metastasis (p = 0.05). Meanwhile, AdipoR2 immunohistochemical stain expression was inversely correlated with T-stage (p = 0.05) and AdipoR2 mRNA expression was significantly lower in patients with smoker's bronchiolitis (p = 0.01) and emphysema (p = 0.03). Patients with expression of AdipoR1 had longer overall survival. AdipoR2 expression was not correlated with patients' survival. In conclusion, we suggest that expression of AdipoR1 is indicative of favorable prognosis and may be used as prognostic marker in NSCLC.

Hydroxynaphthoic acids identified in a high throughput screening potently ameliorate endoplasmic reticulum stress as novel chemical chaperones.

Folding of newly synthesized protein occurs in endoplasmic reticulum (ER) and is assisted by chaperone molecules. In ER stress conditions, misfolded proteins are enriched in a lumen of ER perturbing its normal function, which triggers cellular self-defense mechanism, the unfolded protein response (UPR). It was reported that tunicamycin-induced ER stress can be modulated with high concentration of chemicals such as 4-phenylbutyric acid and salicylate. In search of assay systems to identify such compounds, we have developed a cell-based reporter assay where renilla luciferase activity is driven by glucose-regulated protein 78 (GRP78) promoter. Using our reporter assay, we have screened chemical libraries and found that hydroxynaphthoic acids, especially 1-, 3-, and 6-hydroxy-2-naphthoic acids, potently decrease the ER stress signal, showing an order of magnitude better activity than salicylate. UPR markers such as GRP78, C/EBP homology protein (CHOP) and phosphorylated protein kinase RNA-activated (PKR)-like ER kinase (PERK) were significantly down-regulated with hydroxynaphthoic acids in western blot. Among the analogues, 1-hydroxy-2-naphthoic acid was the most potent in down-regulating those UPR markers. Further, both phosphorylated inositol-requiring enzyme 1α (IRE1α) and spliced form of X-box binding protein 1 (XBP1) were decreased in the protein and the mRNA level, implying both PERK and IRE1α branches in UPR mechanism are controlled with hydroxynaphthoic acids. Taken together, it was suggested that hydroxynaphthoic acids exert their ER stress-reducing activity prior to the UPR activation as chemical chaperones do. In summary, we report a cell-based assay system for the screening of ER stress-reducing compounds and hydroxynaphthoic acids as novel series of chemical chaperones.

Comparative Analysis of Driver Mutations and Transcriptomes in Papillary Thyroid Cancer by Region of Residence in South Korea.

BACKGRUOUND: Radiation exposure is a well-known risk factor for papillary thyroid cancer (PTC). South Korea has 24 nuclear reactors in operation; however, no molecular biological analysis has been performed on patients with PTC living near nuclear power plants. METHODS: We retrospectively included patients with PTC (n=512) divided into three groups according to their place of residence at the time of operation: inland areas (n=300), coastal areas far from nuclear power plants (n=134), and nuclear power plant areas (n=78). After propensity score matching (1:1:1) by age, sex, and surgical procedure, the frequency of representative driver mutations and gene expression profiles were compared (n=50 per group). Epithelial-mesenchymal transition (EMT), BRAF, thyroid differentiation, and radiation scores were calculated and compared. RESULTS: No significant difference was observed in clinicopathological characteristics, including radiation exposure history and the frequency of incidentally discovered thyroid cancer, among the three groups. BRAFV600E mutation was most frequently detected in the groups, with no difference among the three groups. Furthermore, gene expression profiles showed no statistically significant difference. EMT and BRAF scores were higher in our cohort than in cohorts from Chernobyl tissue bank and The Cancer Genome Atlas Thyroid Cancer; however, there was no difference according to the place of residence. Radiation scores were highest in the Chernobyl tissue bank but exhibited no difference according to the place of residence. CONCLUSION: Differences in clinicopathological characteristics, frequency of representative driver mutations, and gene expression profiles were not observed according to patients' region of residence in South Korea.

Immune response and mesenchymal transition of papillary thyroid carcinoma reflected in ultrasonography features assessed by radiologists and deep learning.

INTRODUCTION: Ultrasonography (US) features of papillary thyroid cancers (PTCs) are used to select nodules for biopsy due to their association with tumor behavior. However, the molecular biological mechanisms that lead to the characteristic US features of PTCs are largely unknown. OBJECTIVES: This study aimed to investigate the molecular biological mechanisms behind US features assessed by radiologists and three convolutional neural networks (CNN) through transcriptome analysis. METHODS: Transcriptome data from 273 PTC tissue samples were generated and differentially expressed genes (DEGs) were identified according to US feature. Pathway enrichment analyses were also conducted by gene set enrichment analysis (GSEA) and ClusterProfiler according to assessments made by radiologists and three CNNs - CNN1 (ResNet50), CNN2 (ResNet101) and CNN3 (VGG16). Signature gene scores for PTCs were calculated by single-sample GSEA (ssGSEA). RESULTS: Individual suspicious US features consistently suggested an upregulation of genes related to immune response and epithelial-mesenchymal transition (EMT). Likewise, PTCs assessed as positive by radiologists and three CNNs showed the coordinate enrichment of similar gene sets with abundant immune and stromal components. However, PTCs assessed as positive by radiologists had the highest number of DEGs, and those assessed as positive by CNN3 had more diverse DEGs and gene sets compared to CNN1 or CNN2. The percentage of PTCs assessed as positive or negative concordantly by radiologists and three CNNs was 85.6% (231/273) and 7.1% (3/273), respectively. CONCLUSION: US features assessed by radiologists and CNNs revealed molecular biologic features and tumor microenvironment in PTCs.

Design and Development of IKZF2 and CK1α Dual Degraders.

Lenalidomide achieves its therapeutic efficacy by recruiting and removing proteins of therapeutic interest through the E3 ligase substrate adapter cereblon. Here, we report the design and characterization of 81 cereblon ligands for their ability to degrade the transcription factor Helios (IKZF2) and casein kinase 1 alpha (CK1α). We identified a key naphthamide scaffold that depleted both intended targets in acute myeloid leukemia MOLM-13 cells. Structure-activity relationship studies for degradation of the desired targets over other targets (IKZF1, GSPT1) afforded an initial lead compound DEG-35. A subsequent scaffold replacement campaign identified DEG-77, which selectively degrades IKZF2 and CK1α, and possesses suitable pharmacokinetic properties, solubility, and selectivity for in vivo studies. Finally, we show that DEG-77 has antiproliferative activity in the diffuse large B cell lymphoma cell line OCI-LY3 and the ovarian cancer cell line A2780 indicating that the dual degrader strategy may have efficacy against additional types of cancer.

Dual IKZF2 and CK1α degrader targets acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a hematologic malignancy for which several epigenetic regulators have been identified as therapeutic targets. Here we report the development of cereblon-dependent degraders of IKZF2 and casein kinase 1α (CK1α), termed DEG-35 and DEG-77. We utilized a structure-guided approach to develop DEG-35 as a nanomolar degrader of IKZF2, a hematopoietic-specific transcription factor that contributes to myeloid leukemogenesis. DEG-35 possesses additional substrate specificity for the therapeutically relevant target CK1α, which was identified through unbiased proteomics and a PRISM screen assay. Degradation of IKZF2 and CK1α blocks cell growth and induces myeloid differentiation in AML cells through CK1α-p53- and IKZF2-dependent pathways. Target degradation by DEG-35 or a more soluble analog, DEG-77, delays leukemia progression in murine and human AML mouse models. Overall, we provide a strategy for multitargeted degradation of IKZF2 and CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay.

To identify potent antiviral compounds, we introduced a high-throughput screen platform that can rapidly classify hit compounds according to their target. In our platform, we performed a compound screen using a lentivirus-based pseudovirus presenting a spike protein of coronavirus, and we evaluated the hit compounds using an amplified luminescence proximity homogeneous assay (alpha) test with purified host receptor protein and the receptor binding domain of the viral spike. With our screen platform, we were able to identify both spike-specific compounds (class I) and broad-spectrum antiviral compounds (class II). Among the hit compounds, thiosemicarbazide was identified to be selective to the interaction between the viral spike and its host cell receptor, and we further optimized the binding potency of thiosemicarbazide through modification of the pyridine group. Among the class II compounds, we found raloxifene and amiodarone to be highly potent against human coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2. In particular, using analogs of the benzothiophene moiety, which is also present in raloxifene, we have identified benzothiophene as a novel structural scaffold for broad-spectrum antivirals. This work highlights the strong utility of our screen platform using a pseudovirus assay and an alpha test for rapid identification of potential antiviral compounds and their mechanism of action, which can lead to the accelerated development of therapeutics against newly emerging viral infections.

Proteomics-based strategy to delineate the molecular mechanisms of RhoGDI2-induced metastasis and drug resistance in gastric cancer.

Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.

Let-7 modulates acquired resistance of ovarian cancer to Taxanes via IMP-1-mediated stabilization of multidrug resistance 1.

Ovarian cancer patients frequently develop resistance to chemotherapy regiments using Taxol and carboplatin. One of the resistance factors that protects cancer cells from Taxol-based therapy is multidrug resistance 1 (MDR1). micro(mi)RNAs are small noncoding RNAs that negatively regulate protein expression. Members of the let-7 family of miRNAs are downregulated in many human cancers, and low let-7 expression has been correlated with resistance to microtubule targeting drugs (Taxanes), although little is known that would explain this activity. We now provide evidence that, although let-7 is not a universal sensitizer of cancer cells to Taxanes, it affects acquired resistance of cells to this class of drugs by targeting IMP-1, resulting in destabilization of the mRNA of MDR1. Introducing let-7g into ADR-RES cells expressing both IMP-1 and MDR1 reduced expression of both proteins rendering the cells more sensitive to treatment with either Taxol or vinblastine without affecting the sensitivity of the cells to carboplatin, a non-MDR1 substrate. This effect could be reversed by reintroducing IMP-1 into let-7g high/MDR1 low cells causing MDR1 to again become stabilized. Consistently, many relapsed ovarian cancer patients tested before and after chemotherapy were found to downregulate let-7 and to co-upregulate IMP-1 and MDR1, and the increase in the expression levels of both proteins after chemotherapy negatively correlated with disease-free time before recurrence. Our data point at IMP-1 and MDR1 as indicators for response to therapy, and at IMP-1 as a novel therapeutic target for overcoming multidrug resistance of ovarian cancer.