Kaempferol attenuates viability of ex-vivo cultured post-NACT breast tumor explants through downregulation of p53 induced stemness, inflammation and apoptosis evasion pathways.

Early onset of chemotherapy evasion is a therapeutic challenge. Chemotherapy-induced upregulation of stem cell markers imparts invasiveness and metastatic property to the resident tumor. The efficacy of Kaempferol in attenuating epithelial to mesenchymal transition has earlier been established in the breast cancer cell. In our study population, progression-free survival was observed to be statistically more significant in post-NACT low-grade tumors than the high-grade tumors. Further, in post-NACT TNBCs, high-grade tumors showed a preponderance of strong nuclear p53 expression and very low expression of Caspase 3, indicating that, altered p53 expression predisposes these tumors to apoptosis escape and up-regulation of stemness markers. Herein, we report the robust efficacy of Kaempferol on ex-vivo grown breast tumors, derived from post-NACT TNBC patients, through downregulation of nuclear p53, CD44, ALDH1, NANOG, MDR1, Ki67, BCL2 and upregulation of Caspase 3. Such tumors also showed concurrent deregulated RNA and protein expression of CD44, NANOG, ALDH1 and MDR1 with upregulation of Caspase 3 and cleaved Caspase 3, upon Kaempferol treatment. Validation of efficacy of the treatment dosage of Kaempferol through immunophenotyping on MDA-MB-231, suggested that Kaempferol at its IC-50 dosage was effective against CD44 and CD326 positive breast cancer through deregulating their expression. Protein-protein interaction network through STRING pathway analysis and co-expression study of candidate proteins showed the highest degree of co-expression of p53 and KI-67, CD44, NF- kappaB, ALDH1, NANOG, MDR1, and BCL2. Thus, potentially targetable oncogenic protein markers, that are susceptible to downregulation by Kaempferol, provides insight into biomarker-driven therapeutic approaches with it.

HIWI2 induces G2/M cell cycle arrest and apoptosis in human fibrosarcoma via the ROS/DNA damage/p53 axis.

AIMS: Piwi-like RNA-mediated gene silencing 4 (PIWIL4) or HIWI2, are seen deregulated in human cancers and possibly play critical roles in tumorigenesis. It is unknown what role HIWI2 plays in the regulation of fibrosarcoma, an early metastatic lethal type of soft tissue sarcoma (STS). The present study aimed to investigate the role of HIWI2 in the tumorigenesis of fibrosarcoma. MAIN METHODS: The expression of HIWI2 in HT1080 fibrosarcoma cells was determined by qRT-PCR and western blotting. The MTT assay, colony formation assay, cell cycle, and PE-AnnexinV/7AAD apoptosis assay using flow cytometry, DNA laddering assay, comet assay, and γH2AX accumulation assay were performed to study the effect of HIWI2 overexpression in HT1080 cells. Further, the effect of silencing of HIWI2 was determined by cell viability assay, transwell migration, and invasion assay. KEY FINDINGS: HIWI2 is under-expressed in STS cell lines and tissues, which is associated with poor disease-free survival, disease-specific survival, and progression-free survival of the patients. Overexpression of HIWI2 in HT1080 cells causes DNA damage by increasing intracellular ROS by inhibiting the expression of antioxidant genes (SOD1, SOD2, GPX1, GPX4, and CAT). Furthermore, an increase in H2AX phosphorylation was observed, which activates p53 that promotes p21 expression and caspase-3 activation, leading to G2/M phase cell cycle arrest and apoptosis. HIWI2 silencing, on the contrary, promotes cell growth, migration, and invasion by activating MMP2 and MMP9. SIGNIFICANCE: These results are the first to show that HIWI2 acts as a tumor suppressor in fibrosarcoma by modulating the ROS/DNA damage/p53 pathway.

miR-197-5p increases Doxorubicin-mediated anticancer cytotoxicity of HT1080 fibrosarcoma cells by decreasing drug efflux.

Doxorubicin (Dox) is one of the most used drugs in the treatment of Soft tissue sarcoma. However, acquired resistance linked with poor survival and numerous side effects are the major challenges. Meanwhile, miRNAs are reported to influence the chemotherapeutic responses. However, there is hardly any evidence on the involvement of tumor-suppressive miR-197 reported in our previous study in augmenting the sensitivity of fibrosarcoma cells to Dox. Therefore, in this study, we intend to decipher if miR-197-5p combined with Dox could increase the anticancer cytotoxicity. For this, we evaluated the antitumorigenic effects of Dox and miR-197-5p individually and in combination by performing a series of molecular assays. We noticed that the sub-lethal concentration of miR-197-5p markedly enhanced the sensitivity of HT1080 fibrosarcoma cells to Dox by promoting apoptosis and G2/M cell cycle arrest. We also observed miR-197-5p sensitizes HT1080 cells to Dox by increasing drug influx, possibly due to suppression of MDR genes (ABCC1, MVP). Moreover, we found that KIAA0101, a target of miR-197-5p is inhibited by Dox, which is further repressed when treated in combination with miRNA. We also observed a marked upregulation of p53, known to be negatively correlated with KIAA0101 in Dox and miR-197-5p combination treatment compared to Dox alone. Taken together, our study revealed that Dox chemotherapy in combination with miR-197-5p could overcome the problem of drug efflux and enhance its antitumor effects on fibrosarcoma.

Ribonucleotide reductase subunit M2 is a potential prognostic marker and therapeutic target for soft tissue sarcoma.

Soft tissue sarcomas (STSs) are highly aggressive malignant tumors that exhibit poor therapeutic outcomes. Hence, we aimed to track down a potential gene that can be used as a prognostic marker and therapeutic target for this malignancy. We integrated omics analysis of clinical data and in vitro studies and identified Ribonucleotide reductase subunit M2 (RRM2) as a potential oncogene associated with STS prognosis. We found RRM2 is highly expressed in STS cell lines and tissues. STS patients with increased RRM2 levels showed worse overall survival, disease-free survival, progression-free survival, and disease-specific survival. Further, overexpression of RRM2 in HT1080 cells induces proliferation, migration, invasion, and colony formation, whereas its silencing arrest the cell cycle at G0/G1 phase and induces apoptosis. Taken together, we established RRM2 to be positively associated with oncogenesis and prognosis of STS and therefore could be a promising prognostic marker and therapeutic target.

piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair.

Resistance to doxorubicin (DOX) is an obstacle to successful sarcoma treatment and a cause of tumor relapse, with the underlying molecular mechanism still unknown. PIWI-interacting RNAs (piRNAs) have been shown to enhance patient outcomes in cancers. However, there are few or no reports on piRNAs affecting chemotherapy in cancers, including fibrosarcoma. The current study aims to investigate the relationship between piR-39980 and DOX resistance and the underlying mechanisms. We reveal that piR-39980 is less expressed in DOX-resistant HT1080 (HT1080/DOX) fibrosarcoma cells. Our results show that inhibition of piR-39980 in parental HT1080 cells induces DOX resistance by attenuating intracellular DOX accumulation, DOX-induced apoptosis, and anti-proliferative effects. Its overexpression in HT1080/DOX cells, on the other hand, increases DOX sensitivity by promoting intracellular DOX accumulation, DNA damage, and apoptosis. The dual-luciferase reporter assay indicates that piR-39980 negatively regulates RRM2 and CYP1A2 via direct binding to their 3'UTRs. Furthermore, overexpressing RRM2 induces DOX resistance of HT1080 cells by rescuing DOX-induced DNA damage by promoting DNA repair, whereas CYP1A2 confers resistance by decreasing intracellular DOX accumulation, which piR-39980 restores. This study reveals that piR-39980 could reduce fibrosarcoma resistance to DOX by modulating RRM2 and CYP1A2, implying that piRNA can be used in combination with DOX.

Restoration of microRNA-197 expression suppresses oncogenicity in fibrosarcoma through negative regulation of RAN.

MicroRNAs (miRNAs) act as crucial regulators of biological pathways/processes by reinforcing transcriptional programs and moderating transcripts. Emerging evidences have shown the involvement of dysregulated miRNAs in pathophysiology of human diseases including several cancer types. Recently, miR-197-3p has been reported to play different roles in different cancers; however, its role in fibrosarcoma, a highly aggressive and malignant soft tissue sarcoma originated from the mesenchymal tissues, has not yet been studied. Therefore, this study aims to investigate the possible regulatory roles of miR-197-3p in the oncogenicity of fibrosarcoma. For this, we initially performed qRT-PCR of miR-197-3p, which we found to be downregulated in HT1080 human fibrosarcoma cells compared with IMR90-tert normal fibroblast cells. Subsequently, we performed gain-of-function study by employing several methods such as MTT assay, clonogenic assay, wound healing, flow cytometry cell cycle analysis, and acridine orange staining after transfecting HT1080 cells with miR-197-3p mimic. From these assays, we observed that miR-197-3p significantly inhibits viability, colony forming, and migration ability as well as triggers G2/M phase cell cycle arrest and autophagy in fibrosarcoma cells. To understand the mechanism through which miRNA performs these functions, we predicted its targets using TargetScan and performed pathway enrichment analysis after screening them by their expression in fibrosarcoma. Among the enriched targets, we found RAN (ras-related nuclear protein) to be a crucial target through which miR-197-3p represses tumorigenesis by binding to its 3´ UTR, validated by luciferase reporter assay. The tumor suppressive role of the miRNA was further confirmed by transfecting its mimic in RAN-overexpressed cells which showed significant attenuation in tumorigenic effect of RAN in fibrosarcoma as seen in different assays. Taken together, our study unveiled that miR-197-3p acts as an oncosuppressor in fibrosarcoma through G2/M phase arrest and induction of autophagy, and raises the possibility to act as a novel therapeutic intervention for the malignancy.

piR-39980 promotes cell proliferation, migration and invasion, and inhibits apoptosis via repression of SERPINB1 in human osteosarcoma.

BACKGROUND INFORMATION: Piwi-interacting RNAs (piRNAs) are a novel class of ∼23-36 nts long endogenous small non-coding RNAs, earlier known to maintain germline genome integrity during development by regulating transposable elements. Recently, piRNAs are known to regulate cell proliferation, apoptosis and metastasis in different cancer cells. However, piRNAs have not yet been reported in human osteosarcoma (OS), a highly metastatic aggressive bone tumour among adolescents. Therefore, our current study aimed to decrypt the potential role of a piRNA, piR-39980 in osteosarcoma, which was earlier reported by us in fibrosarcoma, neuroblastoma and epithelial ovarian cancer. RESULTS: We found that piR-39980 is significantly upregulated in two human osteosarcoma cell lines, 143B and HOS compared to IMR90-tert fibroblast cells. The transient overexpression of endogenous piR-39980 level through transfection by piRNA mimic promotes proliferation, migration and invasion ability of OS cells, whereas its inhibition significantly induces apoptosis, chromatin condensation and γ-H2AX accumulation as well as restrains migration and invasion of OS cells. Further, we found 13 genes as targets of piR-39980 using miRanda, among which SERPINB1 that is downregulated in OS cells is seen to suppress proliferation, and migration in this cancer upon its overexpression. The knockdown of piR-39980 in OS cells led to enhanced expression of SERPINB1 indicating to be its target, which was then confirmed by dual luciferase reporter assay. In addition, gelatin zymography and western blotting revealed that transfection of piR-39980 mimic promotes MMP-2 activation, whereas its inhibition and SERPINB1 overexpression suppresses MMP-2 activation in OS cells. CONCLUSIONS: Taken together, our study revealed that piR-39980 promotes migration and invasion via MMP-2 activation as well as inhibits cell death, both through negative regulation of SERPINB1. SIGNIFICANCE: This study revealed that piR-39980 functions as an oncogene in human osteosarcoma, which could be harnessed as a potential therapeutic target for this malignancy.

PIWI-interacting RNA 39980 promotes tumor progression and reduces drug sensitivity in neuroblastoma cells.

Neuroblastoma (NB) is the leading pediatric cancer known for its heterogeneity and clinical aggressiveness leading to chemoresistance. Recent evidence in small RNA research has led to the discovery of PIWI-interacting RNAs (piRNAs) which work in an orchestrated fashion to modulate gene expression both in homeostatic conditions and abnormalities like cancer including NB. This study aims to decipher the possible role of a repeat-derived piRNA, piR-39980 (identified from our previous piRNA profiling study in human NB cell lines) in tumorigenesis of NB cells. piR-39980, overexpressed in NB cells act as an oncopiR and promotes tumor progression, while its inhibition resulted in reduced viability, invasion as well as the migration of IMR-32 cells. Interestingly, we observed that inhibition of piRNA induces senescence of NB cells without affecting the classical apoptosis pathway by modulating the expression of JAK3 through target binding. In addition, piR-39980 was found to desensitize the effect of doxorubicin and inhibit drug-induced apoptosis. Overall, we report piR-39980, as the first oncopiR which might serve as a novel therapeutic target for this malignancy.

miR-197-5p inhibits sarcomagenesis and induces cellular senescence via repression of KIAA0101.

The abnormal expressions of microRNAs (miRNAs) are known to be associated with various pathophysiological processes that lead to the development of a plethora of diseases including cancer. Among several miRNAs studied so far, miR-197 has been reported to play a vital role either as an oncogene or tumor suppressor in different cancers. However, its role in carcinogenesis of fibrosarcoma has not yet been elucidated. Therefore, the current study investigated the role of miR-197-5p, which is significantly downregulated in HT1080 fibrosarcoma cells compared to IMR90-tert fibroblast cells. The transient overexpression of miR-197-5p causes a significant decrease in viability and proliferation of fibrosarcoma cells in both concentration- and time-dependent manners. Interestingly, we did not observe any significant changes in cell cycle pattern or apoptotic cell populations, but rather noticed cellular senescence of fibrosarcoma cells upon overexpression of miR-197-5p. Further, this miRNA suppresses the metastatic properties, such as migration, invasion, and anchorage-independent growth of fibrosarcoma possibly through targeting KIAA0101, which is a proliferating cell nuclear antigen-associated factor and overexpressed in the malignancy. In nutshell, our result revealed that miR-197-5p acts as an oncosuppressor miRNA in fibrosarcoma through target regulation of KIAA0101, which can be exploited for developing RNA-based therapeutic strategies for the cure of this malignancy.

Tumor suppressive activity of PIWI-interacting RNA in human fibrosarcoma mediated through repression of RRM2.

P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a promising class of small regulatory RNAs, earlier believed to control transposable elements (TEs) activity in germlines are now reported in somatic and cancer cells. The aberrant expression of piRNAs has been documented in various cancers wherein they modulate tumorigenesis either as oncogenes or tumor suppressors by curbing target gene expression. However, there is no report yet on the association of piRNAs in fibrosarcoma, an early metastatic lethal tumor. For the first time, we reported a piRNA, piR-39980 in fibrosarcoma and investigated its potential role in malignancy by employing several methods such as qRT-PCR, MTT assay, transwell invasion and migration assay, wound healing assay, flow cytometric cell cycle analysis, Annexin V-PE apoptosis assay, AO/EB dual staining assay, and chromatin condensation assay. We observed that piR-39980 significantly attenuated proliferation, migration, invasion, and colony forming ability as well as induced apoptotic cell death of HT1080 fibrosarcoma cells when transiently overexpressed with its piRNA mimics. The dual luciferase reporter assay confirmed that piR-39980 promotes apoptosis and inhibits proliferation in fibrosarcoma by repressing RRM2 through direct targeting at its 3'UTR through extensive sequence complementary binding, unlike microRNA targeting. In summary, this study revealed that piR-39980 has a strong anti-tumor effect and hence could be a promising RNA-based therapeutic agent for the malignancy of fibrosarcoma.

Preferential binding to zinc oxide nanoparticle interface inhibits lysozyme fibrillation and cytotoxicity.

The aim of present investigation is to explore the effect of zinc oxide nanoparticles (ZnONP, 30 nm) interface on conformational dynamics and stability of lysozyme, at pH 7.4 and pH 9.0. Lysozyme adopts partially disordered conformation at pH 9.0, which adopts fibril morphology in presence of sodium dodecyl sulfate (SDS), compared to the conformation adopted at pH 7.4. However, the presence of ZnONP interface renders partially disordered lysozyme relatively regular and non-amyloidogenic conformation, and enhances the functional efficacy of lysozyme at pH 9.0. Additionally, the thermograms reveal a non-cooperative unfolding of the pH 9.0 lysozyme conformation, which accompanied with intermediate conformations that increased with increase in the interface concentration. The binding thermodynamics indicate that at pH 9.0, lysozyme conformation preferentially binds to ZnONP interface than SDS interface. The preferential binding is attributed for the resulting anti-fibrillation propensity of ZnONP interface. The data, altogether, suggest that the presence of ZnONP interface resulted in conformational rearrangements in the partially disordered lysozyme at pH 9.0 causing accumulation of non-amyloidogenic and functionally active intermediates, thus shielding the lysozyme from SDS induced fibrillation and cytotoxicity.