Cellular activity of microRNAs dysregulated in breast cancer

Breast cancer is one of the major health problems worldwide and it is the second cause of cancer-related in women. Patients often develop resistance to the current therapies. For this reason, the identification of new specific clinical molecular markers and pharmacologic targets in cancer research is an ongoing challenge. Over the last years, microRNAs (miRNAs) have become one of the main subjects of study in the area of cancer genomics. They negatively regulate gene expression post-transcriptional by inhibiting translation and causing degradation of target mRNA. More than a thousand miRNAs exist in the human genome and each one can potentially regulate hundreds of mRNAs. By regulating the expression of target genes, miRNAs can have a tumor suppressor or oncogenic role. Therefore, miRNAs can play an important role in all the phases of cancer, like as initiation, progression, growth, apoptosis, invasion and metastasis. In a previous study based on miRNA profiling, in different solid tumors, comprised breast cancer, and normal tissues, several miRNA were reported to be over- or down-regulated in solid tumors in comparison to normal tissues (Volinia et al. Genome Res. 2010). In other scientific reports other miRNAs were positively or negatively correlated with tumor progression (Volinia S. and Croce CM. PNAS 2013). Starting from these literature data, we decided to analyze the in vitro effect of the administration of this tumor-related miRNAs in order to verify their effect on the viability and transcriptional regulation in breast cancer cell line, in order to gain experimental evidences on their actual involvement in the tumor illness. Firstly, we have chosen 10 different cell lines of breast cancer origin (MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-361, SKBR3, T47D, BT474, ZR75.1, MDA-MB-453, HBL-100) and on 2 cell lines of breast normal epithelium (MCF10A and 184A1). Genomic analysis revealed presence of a complex panel of cancer-related mutations, and the cell lines were different in term of cell growth. We checked also the miRNA levels inside cell lines. Two groups of primary solid tumor-related (23 miRNAs: hsa-miR-126*, hsa-let-7d*, hsa-miR-326, hsa-miR-320c, hsa-miR-302a, hsa-miR-222, hsa-miR-218, hsa-miR-210, hsa-miR-206, hsa-miR-203, hsa-miR-202, hsa-miR-181a, hsa-miR-142, hsa-miR-145, hsa-miR-143, hsa-miR-138, hsa-miR-130b, hsa-miR-126, hsa-miR-99a, hsa-miR-28-5p, hsa-miR-33b, hsa-miR-26b, hsa-miR-21) or progression-related (15 miRNAs: hsa-miR-9, hsa-miR-10a, hsa-miR-25, hsa-miR-27, hsa-miR-30a, hsa-miR-93, hsa-miR-103, hsa-miR-148b, hsa-miR-151, hsa-miR-301a, hsa-miR-328, hsa-miR-484, hsa-miR-615, hsa-miR-874, hsa-miR-1307) miRNAs were transiently transfected into cells, and viability was assessed. We were able to experimentally identify two groups of miRNAs which were able to significantly increase or decrease cell viability. The miRNAs that showed to increase cellular viability in five out twelve cell lines were: miR-130b, miR-138, miR-210, miR-148b and miR-1307. On the other hand, miR-93, miR-126 and miR-145 displayed a significant inhibitory effect on cell viability in five out twelve cell lines. These miRNAs were further investigated for their capacity to affect cell migration, cell invasion, and genome profiling. The main outcome of our work has been the identification from a wide list of cancer-related miRNAs of few of them involved in the in vitro regulation of cell growth and invasion. As a first attempt to identify target genes commonly regulated in vivo and in vitro, we have bioinformatically identified, in a first not exhaustive screening, PTEN and DICER1 genes, which in vivo and in vitro negatively correlated with miR-210 and miR-130b, respectively

Loss of miR-204 expression is a key event in melanoma

Cutaneous melanoma (CM) is a malignancy with increasing occurrence. Its microRNA repertoire has been defined in a number studies, leading to candidates for biological and clinical relevance: miR-200a/b/c, miR-203, miR-205, miR-204, miR-211, miR-23b and miR-26a/b. Our work was aimed to validate the role of these candidate miRNAs in melanoma, using additional patients cohorts and in vitro cultures. miR-26a, miR-204 and miR-211 were more expressed in normal melanocytes, while miR-23b, miR-200b/c, miR-203 and miR-205 in epidermis and keratinocytes. None of the keratinocyte-related miRNAs was associated with any known mutation or with clinical covariates in melanoma. On the other hand, the loss of miR-204 was enriched in melanomas with NRAS sole mutation (Fisher exact test, P = 0.001, Log Odds = 1.67), and less frequent than expected in those harbouring CDKN2A mutations (Fisher exact test, P = 0.001, Log Odds − 1.09). Additionally, miR-204 was associated with better prognosis in two independent melanoma cohorts and its exogenous expression led to growth impairment in melanoma cell lines. Thus, miR-204 represents a relevant mechanism in melanoma, with potential prognostic value and its loss seems to act in the CDKN2A pathway, in cooperation with NRAS

Synergistic cytotoxic activity of recombinant TRAIL plus the non-genotoxic activator of the p53 pathway nutlin-3 in acute myeloid leukemia cells

To potentiate the response of acute myeloid leukemia (AML) to TRAIL cytotoxicity, we have adopted a strategy of combining nutlin-3, a potent non-genotoxic activator of the p53 pathway, with recombinant TRAIL. The rationale for using such a combination was that deletions and/or mutations of the p53 gene occur in only 5-10% of AML and that TRAIL and nutlin-3 activate the extrinsic and intrinsic pathways of apoptosis, respectively. TRAIL induced a rapid increase of apoptosis when added to OCI M4-type and MOLM M5-type AML cells, carrying a wild-type p53, as well as to NB4 M3-type AML, carrying a mutated p53. On the other hand, the small molecule activator of the p53 pathway nutlin-3 induced p53 accumulation, cell cycle arrest and a slow progressive increase of apoptosis in OCI and MOLM but not in NB4. Of note, nutlin-3 up-regulated the surface expression of TRAIL-R2 and synergized with TRAIL in inducing apoptosis in OCI and MOLM as well as in primary M4-type and M5-type AML blasts, but not in NB4 cells. Moreover, while nutlin-3 up-regulated the expression of cyclin dependent kinase inhibitor p21, a p53-target gene mediating cell cycle block and showing anti-apoptotic activity, the simultaneous addition of TRAIL plus nutlin-3 induced the caspase-dependent cleavage of p21. The relevance of p21 down-regulation for sensitizing AML cells to apoptosis was underscored in knocking-down experiments with small interfering RNAs. Our data suggest that the combined treatment of nutlin-3 plus TRAIL might offer a novel therapeutic strategy for AM

Screen for MicroRNA and Drug Interactions in Breast Cancer Cell Lines Points to miR-126 as a Modulator of CDK4/6 and PIK3CA Inhibitors

Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR).Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann–Whitney U-test with Bonferroni correction was used for statistical analysis.Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC50 was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2+). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis.Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation

Calmodulin expression distinguishes the smooth muscle cell population of human carotid plaque

Several observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset in atherosclerosis and restenosis. We characterized the phenotypic features of SMC subsets in cultures derived from human carotid endarterectomy specimens. Specimens comprised an undiseased portion (thin intimal thickening with the underlying media) and a diseased portion (atherosclerotic plaque with the underlying media). From plaque tissues of the diseased portion, only macrophage-derived foam cells were retrieved. From medial tissues, two SMC phenotypes were isolated: large SMCs (flat with a monolayered growth pattern, from the undiseased portion) and small SMCs (fusiform and growing in multilayers, from the undiseased and diseased portions after co-culture with macrophage-derived foam cells). Small SMCs displayed higher proliferative and migratory activities and were less differentiated than large SMCs. Proteomic analysis showed that calmodulin was predominant in small SMCs. Co-culture of large SMCs with macrophage-derived foam cells induced a transition to the small phenotype with increased calmodulin expression. The calmodulin inhibitor W-7 decreased the proliferation of small SMCs and prevented the large to small phenotypic transition. In vivo, calmodulin was markedly expressed in SMCs of atherosclerotic plaques and was barely detectable in the media. Macrophage-derived foam cells promote selective migration from the media of atheroma-prone SMCs characterized by calmodulin overexpression. Further studies of small SMCs could be instrumental in understanding atherosclerosis pathogenesis and in planning therapeutic strategies

Image_2_Screen for MicroRNA and Drug Interactions in Breast Cancer Cell Lines Points to miR-126 as a Modulator of CDK4/6 and PIK3CA Inhibitors.PDF

<p>Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR).</p><p>Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann–Whitney U-test with Bonferroni correction was used for statistical analysis.</p><p>Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC₅₀ was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2⁺). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis.</p><p>Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation.</p

Data_Sheet_2_Screen for MicroRNA and Drug Interactions in Breast Cancer Cell Lines Points to miR-126 as a Modulator of CDK4/6 and PIK3CA Inhibitors.XLSX

<p>Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR).</p><p>Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann–Whitney U-test with Bonferroni correction was used for statistical analysis.</p><p>Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC₅₀ was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2⁺). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis.</p><p>Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation.</p

Data_Sheet_1_Screen for MicroRNA and Drug Interactions in Breast Cancer Cell Lines Points to miR-126 as a Modulator of CDK4/6 and PIK3CA Inhibitors.XLSX

<p>Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR).</p><p>Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann–Whitney U-test with Bonferroni correction was used for statistical analysis.</p><p>Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC₅₀ was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2⁺). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis.</p><p>Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation.</p

Table_3_Screen for MicroRNA and Drug Interactions in Breast Cancer Cell Lines Points to miR-126 as a Modulator of CDK4/6 and PIK3CA Inhibitors.PDF

<p>Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR).</p><p>Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann–Whitney U-test with Bonferroni correction was used for statistical analysis.</p><p>Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC₅₀ was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2⁺). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis.</p><p>Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation.</p