Brock Humphries

MicroRNA-200b targets protein kinase Cα and suppresses triple-negative breast cancer metastasis

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and lacks effective targeted therapies. The microRNA-200 (miR-200) family is found to inhibit or promote breast cancer metastasis; however, the underlying mechanism is not well understood. This study was performed to investigate the effect and mechanism of miR-200b on TNBC metastasis and identify targets for developing more efficient treatment for TNBC. We found that miR-200 family expression levels are significantly lower in highly migratory TNBC cells and metastatic TNBC tumors than other types of breast cancer cells and tumors. Ectopically expressing a single member (miR-200b) of the miR-200 family drastically reduces TNBC cell migration and inhibits tumor metastasis in an orthotopic mouse mammary xenograft tumor model. We identified protein kinase Cα (PKCα) as a new direct target of miR-200b and found that PKCα protein levels are inversely correlated with miR-200b levels in 12 kinds of breast cancer cells. Inhibiting PKCα activity or knocking down PKCα levels significantly reduces TNBC cell migration. In contrast, forced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration and tumor metastasis. Further mechanistic studies revealed that PKCα downregulation by miR-200b results in a significant decrease of Rac1 activation in TNBC cells. These results show that loss of miR-200b expression plays a crucial role in TNBC aggressiveness and that miR-200b suppresses TNBC cell migration and tumor metastasis by targeting PKCα. Our findings suggest that miR-200b and PKCα may serve as promising therapeutic targets for metastatic TNBC.

MicroRNAs: Novel Players in Cancer Diagnosis and Therapies

First discovered in 1993, microRNAs (miRNAs) have been one of the hottest research areas over the past two decades. Oftentimes, miRNAs levels are found to be dysregulated in cancer patients. The potential use of miRNAs in cancer therapies is an emerging and promising field, with research finding miRNAs to play a role in cancer initiation, tumor growth, and metastasis. Therefore, miRNAs could become an integral part from cancer diagnosis to treatment in future. This review aims to examine current novel research work on the potential roles of miRNAs in cancer therapies, while also discussing several current challenges and needed future research.

Root-Localized Phytochrome Chromophore Synthesis Is Required for Photoregulation of Root Elongation and Impacts Root Sensitivity to Jasmonic Acid in Arabidopsis

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.

MicroRNA-200b Suppresses Arsenic-transformed Cell Migration by Targeting Protein Kinase Cα and Wnt5b-Protein Kinase Cα Positive Feedback Loop and Inhibiting Rac1 Activation

Background: MiR-200b is able to inhibit tumor cell migration and metastasis, the underlying mechanism is not well understood. Results: PKCα is a new direct target of miR-200b. Conclusion: MiR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and inhibiting Rac1 activation. Significance: This study identifies a new target and mechanism for the inhibitory effect of miR-200b on tumor cell migration. MicroRNA-200b (miR-200b) is a member of miR-200 family that has been found to inhibit cell migration and cancer metastasis; however, the underlying mechanism is not well understood. We previously reported that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells with highly migratory and invasive characteristics, whereas stably re-expressing miR-200b strongly suppresses arsenic-transformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Cα (PKCα) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCα 3′-untranslated region vector luciferase reporter assays, we showed that PKCα is a direct target of miR-200b. Inhibiting PKCα activity or knocking down PKCα expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCα in miR-200b overexpressing cells impaired the inhibitory effect of miR-200b on cell migration. In addition, we also found a positive feedback loop between Wnt5b and PKCα in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCα expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCα increased Wnt5b and phospho-PKC levels and cell migration. Further mechanistic studies revealed that Rac1 is highly activated in arsenic-transformed cells and stably expressing miR-200b abolishes Rac1 activation changing actin cytoskeleton organization. Manipulating PKCα or Wnt5b expression levels significantly altered the level of active Rac1. Together, these findings indicate that miR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation.

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin-vascular endothelial growth factor pathway

Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchial epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin-VEGF pathway.

The role of microRNAs in metal carcinogen-induced cell malignant transformation and tumorigenesis.

MicroRNAs (miRNAs), an important component of epigenetic mechanisms of carcinogenesis, have been shown to play crucial roles in cancer initiation, metastasis, prognosis and responses to drug treatment and may serve as biomarkers for early diagnosis of cancer and tools for cancer therapy. Metal carcinogens, such as arsenic, cadmium, hexavalent chromium and nickel, are well-established human carcinogens causing various cancers upon long term exposure. However, the mechanism of metal carcinogenesis has not been well understood, which limits our capability to effectively diagnose and treat human cancers resulting from chronic metal carcinogen exposure. Over recent years, the role of miRNAs in metal carcinogenesis has been actively explored and a growing body of evidence indicates the critical involvement of miRNAs in metal carcinogenesis. This review aims to discuss recent studies showing that miRNAs play important roles in metal carcinogen-induced cell malignant transformation and tumorigenesis. Some thoughts for future further studies in this field are also presented.

ARHGAP18 downregulation by miR-200b suppresses metastasis of triple negative breast cancer by enhancing activation of RhoA

Rho GTPases activated in cancer cells drive proliferation, migration, and metastasis. Thus, RhoGAP proteins, which negatively regulate Rho GTPases, are generally thought to function as tumor suppressors. Here this expectation was challenged by characterization of ARHGAP18, a RhoGAP family member that is selectively overexpressed in highly migratory triple-negative breast cancer (TNBC) cells. In human breast tumors, higher ARHGAP18 levels associated with worse overall survival, recurrence-free survival, and metastasis-free survival. In TNBC cells, ARHGAP18 deletion increased RhoA activation but reduced growth, migration, and metastatic capacity. Mechanistic investigations revealed that ARHGAP18 levels were controlled by miR-200b, the enforced expression of which was sufficient to activate RhoA, enhanced formation of focal adhesions and actin stress fibers, and reduced migration and metastasis. Enforced elevation of ARHGAP18 where miR-200b was stably expressed reduced RhoA activity but increased cell migration. Pharmacologic inhibition of the Rho effector kinase ROCK blocked RhoA signaling and reversed the inhibitory effect of miR-200b on cell migration. Finally, ARHGAP18 overexpression or ROCK inhibition was sufficient to overcome metastatic blockade by miR-200b. Taken together, these results define opposing roles for oncogenic ARHGAP18 and tumor suppressive miR-200b in determining TNBC cell migration and metastatic prowess. Cancer Res; 77(15); 4051-64. ©2017 AACR.

Upregulation of histone-lysine methyltransferases plays a causal role in hexavalent chromium-induced cancer stem cell-like property and cell transformation

ABSTRACT While hexavalent chromium [Cr(VI)] is generally considered as a genotoxic environmental carcinogen, studies showed that Cr(VI) exposure also causes epigenetic changes. However, whether Cr(VI)‐caused epigenetic dysregulations plays an important role in Cr(VI) carcinogenicity remain largely unknown. The aim of this study was to determine if chronic low dose Cr(VI) exposure causes epigenetic changes, the underlying mechanism and whether chronic low dose Cr(VI) exposure‐caused epigenetic dysregulation contributes causally to Cr(VI)‐induced cancer stem cell (CSC)‐like property and cell transformation. Two immortalized human bronchial epithelial cell lines (BEAS‐2B and 16HBE) were exposed to 0.25 &mgr;M of K2Cr2O7 for 20 and 40 weeks to induce cell transformation, respectively. Cr(VI)‐induced epigenetic changes were examined in Cr(VI)‐transformed cells and Cr(VI) exposure‐caused human lung cancer tissues. Pharmacological inhibitors and gene knockdown experiments were used to determine the role of epigenetic dysregulation in Cr(VI) carcinogenicity. We found that chronic Cr(VI) exposure causes epigenetic dysregulation as evidenced by the increased levels of histone H3 repressive methylation marks (H3K9me2 and H3K27me3) and the related histone‐lysing methyltransferases (HMTases). Pharmacological inhibition or knockdown of HMTases reduces H3 repressive methylation marks and malignant phenotypes of Cr(VI)‐transformed cells. Moreover, knockdown of HMTases in parental cells significantly reduces chronic Cr(VI) exposure‐induced CSC‐like property and cell transformation. Further mechanistic study revealed that knockdown of HMTases decreases Cr(VI) exposure‐caused DNA damage. Our findings indicate that chronic Cr(VI) exposure increases H3 repressive methylation marks by increasing the related HMTases expression; and that increased expression of HMTases plays a causal role in Cr(VI)‐induced CSC‐like property and cell transformation. HighlightsChronic low dose Cr(VI) exposure causes epigenetic changes by upregulating HMTasesHMTases upregulation plays a causal role in Cr(VI)‐induced CSC‐like propertyHMTases upregulation plays a causal role in Cr(VI)‐induced cell transformationKnockdown of HMTases reduces Cr(VI)‐caused DNA damage

Functional Isolation of Tumor-Initiating Cells using Microfluidic-Based Migration Identifies Phosphatidylserine Decarboxylase as a Key Regulator

Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses. Using this device, we isolated a small subpopulation of migratory cells with significantly greater tumor formation and metastasis in mouse models. Whole transcriptome sequencing of migratory versus non-migratory cells from two metastatic breast cancer cell lines revealed a unique set of genes as key regulators of tumor-initiating cells. We focused on phosphatidylserine decarboxylase (PISD), a gene downregulated by 8-fold in migratory cells. Breast cancer cells overexpressing PISD exhibited reduced tumor-initiating potential in a high-throughput microfluidic mammosphere device and mouse xenograft model. PISD regulated multiple aspects of mitochondria, highlighting mitochondrial functions as therapeutic targets against cancer stem cells. This research establishes not only a novel microfluidic technology for functional isolation of tumor-initiating cells regardless of cancer type, but also a new approach to identify essential regulators of these cells as targets for drug development.

Nanoparticle-Mediated Therapeutic Agent Delivery for Treating Metastatic Breast Cancer—Challenges and Opportunities

Breast cancer (BC) is the second leading cause of cancer-related death in American women and more than 90% of BC-related death is caused by metastatic BC (MBC). This review stresses the limited success of traditional therapies as well as the use of nanomedicine for treating MBC. Understanding the biological barriers of MBC that nanoparticle in vivo trafficking must overcome could provide valuable new insights for translating nanomedicine from the bench side to the bedside. A view about nanomedicine applied in BC therapy has been summarized with their present status, which is gaining attention in the clinically-applied landscape. The progressions of drug/gene delivery systems, especially the status of their preclinical or clinical trials, are also discussed. Here we highlight that the treatment of metastasis, in addition to the extensively described inhibition of primary tumor growth, is an indispensable requirement for nanomedicine. Along with more innovations in material chemistry and more progressions in biology, nanomedicine will constantly supply more exciting new approaches for targeted drug/gene delivery against MBC.