The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single... more The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24 À/lo CD44 + phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.
Despite significant progress in the treatment of breast cancer, particularly through the use of t... more Despite significant progress in the treatment of breast cancer, particularly through the use of targeted therapy, relapse and chemoresistance remain a major hindrance to the fight to minimize the burden of the disease. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial-to-mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them to reverse EMT and to attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL using MP470 (Amuvatinib) reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with the expression of stem cell genes, regulation of metastases genes, increased tumorigenicity and was important for BCSC invasion and migration. Inactivation of AXL also led to the downregulation of nuclear factor-kB pathway and reduced tumor formation in vivo. Taken together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anticancer therapies and to reduce breast cancer recurrence and metastases.
Supplementary Figure Legends 1-5, Methods from Immune-Induced Epithelial to Mesenchymal Transitio... more Supplementary Figure Legends 1-5, Methods from Immune-Induced Epithelial to Mesenchymal Transition In vivo Generates Breast Cancer Stem Cells
Little is known as to whether there may be any pathogenetic link between pulmonary carcinoids and... more Little is known as to whether there may be any pathogenetic link between pulmonary carcinoids and neuroendocrine carcinomas. An eight-gene signature with copy number variations (CNVs) in neuroendocrine neoplasms (NENs), namely MEN1, MYC, MYCL1, RICTOR, RB1, SDHA, SRC and TP53, was used to classify an independent cohort of 54 surgically resectable tumors [31 typical carcinoids (TC), 11 atypical carcinoids (AC) and 12 small cell lung carcinomas (SCLC)], for which transcriptome and mutation data were available. Unsupervised clustering analysis identified two histology-independent clusters, namely CL1 and CL2, where 17/42 (40.5%) carcinoids and all the SCLC samples fell into the latter. CL2 carcinoids affected survival adversely, were enriched in T to G transversions and T>C/C>T transitions in the context of specific mutational signatures, presented with at least 1.5-fold change (FC) increase of gene mutations including TSC2, SMARCA2, SMARCA4, ERBB4 and PTPRZ1, differed for gene e...
Basic & Clinical Pharmacology & Toxicology, 2017
Most FDA-approved drugs are not equally effective in all patients, suggesting that identification... more Most FDA-approved drugs are not equally effective in all patients, suggesting that identification of biomarkers to predict responders to a chemoprevention agent will be needed to stratify patients and achieve maximum benefit. The goal of this study was to investigate both patient-specific and cell context-specific heterogeneity of metformin response, using fibroblast cell lines and induced pluripotent stem cells differentiated into lung epithelial lineages. We performed cell survival analysis, transcriptome and whole exome sequencing analysis on both patient-derived cell lines and cancer cell lines to assess differential metformin response and identify response genes. We found differences in response to metformin treatment across a variety of cell lines and cellular contexts, suggesting that heterogeneity may be patient-and cell type-specific. Gene expression profiling and analysis of metformin-sensitive and metformin-resistant cells identified differentially expressed genes that may be able to stratify patients into metformin responders and non-responders. Sequencing analysis found genomic alterations that correlated with metformin response. These results suggest that the identification of genomic biomarkers for patients who may respond to metformin treatment can provide an opportunity for individualizing metformin chemoprevention in the clinical setting.
Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly ... more Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of αvβ3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo. The TGFβ1/SMAD pathway was identified as a critical signaling axis betw...
The Cancer Genome Atlas Research Network* Adenocarcinoma of the lung is the leading cause of canc... more The Cancer Genome Atlas Research Network* Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.
The development of adenocarcinoma of the lung is believed to proceed from in situ disease (adenoc... more The development of adenocarcinoma of the lung is believed to proceed from in situ disease (adenocarcinoma in situ, AIS) to minimally invasive disease with prominent lepidic growth (minimally invasive adenocarcinoma, MIA), then to fully invasive adenocarcinoma (AD), but direct evidence for this model has been lacking. Because some lung adenocarcinomas show prominent lepidic growth (AD-L), we designed a study to address the lineage relationship between the lepidic (noninvasive) component (L) and the adjacent nonlepidic growth component representing invasive disease within individual tumors. Lineage relationships were evaluated by next-generation DNA sequencing to define large genomic rearrangements in microdissected tissue specimens collected by laser capture. We found a strong lineage relationship between the majority of adjacent lepidic and invasive components, supporting a putative AIS-AD transition. Notably, many rearrangements were detected in the less aggressive lepidic componen...
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, Jan 20, 2014
Distinguishing independent primary tumors from intrapulmonary metastases in non-small-cell carcin... more Distinguishing independent primary tumors from intrapulmonary metastases in non-small-cell carcinoma remains a clinical dilemma with significant clinical implications. Using next-generation DNA sequencing, we developed a chromosomal rearrangement-based approach to differentiate multiple primary tumors from metastasis. Tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal tumors. Laser capture microdissection was performed separately for each tumor. Genomic DNA was isolated using direct in situ whole-genome amplification methodology, and next-generation sequencing was performed using an Illumina mate-pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. A total of 41 tumor samples were sequenced (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]...
The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion... more The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion and/or metastasis in breast carcinomas. However, it is poorly understood how RhoA and RhoC are activated in breast cancer cells. Here we describe the role of myosininteracting guanine nucleotide exchange factor (Myo-GEF) in regulating RhoA and RhoC activation as well as cell polarity and invasion in an invasive breast cancer cell line MDA-MB-231. RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity. The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF. In addition, MyoGEF colocalizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells, and depletion of NMIIA by RNAi disrupts the polarized localization of MyoGEF at the cell leading edge, suggesting a role for NMIIA in regulating MyoGEF localization and function. Moreover, MyoGEF protein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines. Taken together, our results suggest that MyoGEF cooperates with NMIIA to regulate the polarity and invasion activity of breast cancer cells through activation of RhoA and RhoC.
Despite significant progress in the treatment of breast cancer, particularly through the use of t... more Despite significant progress in the treatment of breast cancer, particularly through the use of targeted therapy, relapse and chemoresistance remain a major hindrance to the fight to minimize the burden of the disease. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial-to-mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them to reverse EMT and to attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL using MP470 (Amuvatinib) reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with the expression of stem cell genes, regulation of metastases genes, increased tumorigenicity and was important for BCSC invasion and migration. Inactivation of AXL also led to the downregulation of nuclear factor-kB pathway and reduced tumor formation in vivo. Taken together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anticancer therapies and to reduce breast cancer recurrence and metastases.
Cooperative communications between the central spindle and the contractile ring are critical for ... more Cooperative communications between the central spindle and the contractile ring are critical for the spatial and temporal regulation of cytokinesis. Here we report that MyoGEF, a guanine nucleotide exchange factor that localizes to the central spindle and cleavage furrow, interacts with centrosome/spindle pole-associated protein (CSPP), which is concentrated at the spindle pole and central spindle during mitosis and cytokinesis. Both in vitro and in vivo pulldown assays show that MyoGEF interacts with CSPP. The C-terminus of MyoGEF and N-terminus of CSPP are required for their interaction. Immunofluorescence analysis indicates that MyoGEF and CSPP colocalize at the central spindle. Depletion of CSPP or MyoGEF by RNA-interference (RNAi) not only causes defects in mitosis and cytokinesis, such as metaphase arrest and furrow regression, but also mislocalization of nonmuscle myosin II with a phosphorylated myosin regulatory light chain (p-MRLC). Importantly, CSPP depletion by RNAi inter...
We reported previously that a guanine nucleotide exchange factor, MyoGEF, localizes to the centra... more We reported previously that a guanine nucleotide exchange factor, MyoGEF, localizes to the central spindle, activates RhoA, and is required for cytokinesis. In this study, we have found that Plk1 (polo-like kinase 1) can phosphorylate MyoGEF, thereby recruiting MyoGEF to the central spindle as well as enhancing MyoGEF activity toward RhoA. The in vitro kinase assay shows that Plk1 can phosphorylate MyoGEF on threonine 574. Immunoprecipitation/immunoblot analysis demonstrates that mutation of threonine 574 to alanine dramatically decreases threonine phosphorylation of MyoGEF in transfected HeLa cells, suggesting that threonine 574 is phosphorylated in vivo. Consistent with these observations, immunofluorescence shows that Plk1 and MyoGEF colocalize at the spindle pole and central spindle during mitosis and cytokinesis. Importantly, RNA interference-mediated depletion of Plk1 interferes with the localization of MyoGEF at the spindle pole and central spindle. Moreover, mutation of threonine 574 to alanine in MyoGEF or depletion of Plk1 by RNA interference leads to a decrease in MyoGEF activity toward RhoA in HeLa cells. Therefore, our results suggest that Plk1 can regulate MyoGEF activity and localization, contributing to the regulation of cytokinesis.
Background: Aurora B kinase (aurora B) and polo-like kinase 1 (Plk1) are critical regulators of c... more Background: Aurora B kinase (aurora B) and polo-like kinase 1 (Plk1) are critical regulators of cytokinesis. Results: Phosphorylation of MyoGEF at Thr-544 by aurora B promotes the binding of Plk1 to MyoGEF. Conclusion: Phosphorylation of MyoGEF by aurora B creates a docking site for Plk1. Significance: Coordinated regulation of MyoGEF localization and activation by aurora B and Plk1 contributes to the regulation of cytokinesis. We previously reported that phosphorylation of myosin IIinteracting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.
wuCC5-11-vidS1.mov. Localization of GFPtagged MyoGEF in HeLa cells during cytokinesis. HeLa cells... more wuCC5-11-vidS1.mov. Localization of GFPtagged MyoGEF in HeLa cells during cytokinesis. HeLa cells were transfected with a plasmid encoding GFP-tagged MyoGEF by using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the live HeLa cells expressing GFP-tagged MyoGEF were imaged by using a fluorescence Olympus X170 microscope equipped with a cooled CCD camera.
Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess... more Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial–mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of m...
The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single... more The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell–dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24−/loCD44+ phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have seve...
This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (My... more This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (MyoGEF) and centrosome/spindle pole associated protein (CSPP) in mitotic progression and cytokinesis. We have identified three mouse isoforms of CSPP, all of which interact and colocalize with MyoGEF to the central spindle in anaphase cells. The N-terminus of MyoGEF interacts with myosin whereas the C terminus interacts with the N-terminus of CSPP, forming a complex. The N-terminus of CSPP appears to be important for both localization and interaction with MyoGEF. CSPP plays a role in mitotic progression since its depletion by RNAi resulted in metaphase arrest. MyoGEF is required for completion of cytokinesis. Both MyoGEF and CSPP are phosphorylated by mitotic kinases including Plk1 and Aurora. Importantly, MyoGEF is phosphorylated at Thr-574 in mitosis by Polo-like kinase 1, and this phosphorylation is required for activation of RhoA. Thr-543 of MyoGEF is required for Plk1 binding in mitosis and phosphorylation of MyoGEF by Cdk1/cyclinB, possibly at Thr-543 may generate a Plk1 docking site, i.e., Cdk1 can phosphorylate MyoGEF at Thr-543, thereby allowing Plk1 to bind and phosphorylate MyoGEF at Thr-574. Finally, MyoGEF and CSPP are also phosphorylated by Aurora-B kinase in vitro. Taken together, we propose that Aurora-B may phosphorylate and recruit MyoGEF and CSPP to the central spindle, where phosphorylation of MyoGEF at Thr-543 promotes Polo kinase binding and additional phosphorylation of MyoGEF, leading to the activation of RhoA at the cleavage furrow.
The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single... more The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24 À/lo CD44 + phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.
Despite significant progress in the treatment of breast cancer, particularly through the use of t... more Despite significant progress in the treatment of breast cancer, particularly through the use of targeted therapy, relapse and chemoresistance remain a major hindrance to the fight to minimize the burden of the disease. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial-to-mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them to reverse EMT and to attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL using MP470 (Amuvatinib) reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with the expression of stem cell genes, regulation of metastases genes, increased tumorigenicity and was important for BCSC invasion and migration. Inactivation of AXL also led to the downregulation of nuclear factor-kB pathway and reduced tumor formation in vivo. Taken together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anticancer therapies and to reduce breast cancer recurrence and metastases.
Supplementary Figure Legends 1-5, Methods from Immune-Induced Epithelial to Mesenchymal Transitio... more Supplementary Figure Legends 1-5, Methods from Immune-Induced Epithelial to Mesenchymal Transition In vivo Generates Breast Cancer Stem Cells
Little is known as to whether there may be any pathogenetic link between pulmonary carcinoids and... more Little is known as to whether there may be any pathogenetic link between pulmonary carcinoids and neuroendocrine carcinomas. An eight-gene signature with copy number variations (CNVs) in neuroendocrine neoplasms (NENs), namely MEN1, MYC, MYCL1, RICTOR, RB1, SDHA, SRC and TP53, was used to classify an independent cohort of 54 surgically resectable tumors [31 typical carcinoids (TC), 11 atypical carcinoids (AC) and 12 small cell lung carcinomas (SCLC)], for which transcriptome and mutation data were available. Unsupervised clustering analysis identified two histology-independent clusters, namely CL1 and CL2, where 17/42 (40.5%) carcinoids and all the SCLC samples fell into the latter. CL2 carcinoids affected survival adversely, were enriched in T to G transversions and T>C/C>T transitions in the context of specific mutational signatures, presented with at least 1.5-fold change (FC) increase of gene mutations including TSC2, SMARCA2, SMARCA4, ERBB4 and PTPRZ1, differed for gene e...
Basic & Clinical Pharmacology & Toxicology, 2017
Most FDA-approved drugs are not equally effective in all patients, suggesting that identification... more Most FDA-approved drugs are not equally effective in all patients, suggesting that identification of biomarkers to predict responders to a chemoprevention agent will be needed to stratify patients and achieve maximum benefit. The goal of this study was to investigate both patient-specific and cell context-specific heterogeneity of metformin response, using fibroblast cell lines and induced pluripotent stem cells differentiated into lung epithelial lineages. We performed cell survival analysis, transcriptome and whole exome sequencing analysis on both patient-derived cell lines and cancer cell lines to assess differential metformin response and identify response genes. We found differences in response to metformin treatment across a variety of cell lines and cellular contexts, suggesting that heterogeneity may be patient-and cell type-specific. Gene expression profiling and analysis of metformin-sensitive and metformin-resistant cells identified differentially expressed genes that may be able to stratify patients into metformin responders and non-responders. Sequencing analysis found genomic alterations that correlated with metformin response. These results suggest that the identification of genomic biomarkers for patients who may respond to metformin treatment can provide an opportunity for individualizing metformin chemoprevention in the clinical setting.
Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly ... more Existing antiangiogenic approaches to treat metastatic hepatocellular carcinoma (HCC) are weakly effectual, prompting further study of tumor angiogenesis in this disease setting. Here, we report a novel role for sulfatase 2 (SULF2) in driving HCC angiogenesis. Sulf2-deficient mice (Sulf2 KO) exhibited resistance to diethylnitrosamine-induced HCC and did not develop metastases like wild-type mice (Sulf2 WT). The smaller and less numerous tumors formed in Sulf2 KO mice exhibited a markedly lower microvascular density. In human HCC cells, SULF2 overexpression increased endothelial proliferation, adhesion, chemotaxis, and tube formation in a paracrine fashion. Mechanistic analyses identified the extracellular matrix protein periostin (POSTN), a ligand of αvβ3/5 integrins, as an effector protein in SULF2-induced angiogenesis. POSTN silencing in HCC cells attenuated SULF2-induced angiogenesis and tumor growth in vivo. The TGFβ1/SMAD pathway was identified as a critical signaling axis betw...
The Cancer Genome Atlas Research Network* Adenocarcinoma of the lung is the leading cause of canc... more The Cancer Genome Atlas Research Network* Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.
The development of adenocarcinoma of the lung is believed to proceed from in situ disease (adenoc... more The development of adenocarcinoma of the lung is believed to proceed from in situ disease (adenocarcinoma in situ, AIS) to minimally invasive disease with prominent lepidic growth (minimally invasive adenocarcinoma, MIA), then to fully invasive adenocarcinoma (AD), but direct evidence for this model has been lacking. Because some lung adenocarcinomas show prominent lepidic growth (AD-L), we designed a study to address the lineage relationship between the lepidic (noninvasive) component (L) and the adjacent nonlepidic growth component representing invasive disease within individual tumors. Lineage relationships were evaluated by next-generation DNA sequencing to define large genomic rearrangements in microdissected tissue specimens collected by laser capture. We found a strong lineage relationship between the majority of adjacent lepidic and invasive components, supporting a putative AIS-AD transition. Notably, many rearrangements were detected in the less aggressive lepidic componen...
Journal of clinical oncology : official journal of the American Society of Clinical Oncology, Jan 20, 2014
Distinguishing independent primary tumors from intrapulmonary metastases in non-small-cell carcin... more Distinguishing independent primary tumors from intrapulmonary metastases in non-small-cell carcinoma remains a clinical dilemma with significant clinical implications. Using next-generation DNA sequencing, we developed a chromosomal rearrangement-based approach to differentiate multiple primary tumors from metastasis. Tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal tumors. Laser capture microdissection was performed separately for each tumor. Genomic DNA was isolated using direct in situ whole-genome amplification methodology, and next-generation sequencing was performed using an Illumina mate-pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. A total of 41 tumor samples were sequenced (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]...
The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion... more The small guanine triphosphatase (GTPase) proteins RhoA and RhoC are essential for tumor invasion and/or metastasis in breast carcinomas. However, it is poorly understood how RhoA and RhoC are activated in breast cancer cells. Here we describe the role of myosininteracting guanine nucleotide exchange factor (Myo-GEF) in regulating RhoA and RhoC activation as well as cell polarity and invasion in an invasive breast cancer cell line MDA-MB-231. RNA-interference (RNAi)-mediated depletion of MyoGEF in MDA-MB-231 cells not only suppresses the activation of RhoA and RhoC, but also decreases cell polarity and invasion activity. The dominant-negative mutants of RhoA and RhoC, but not Rac1 and Cdc42, dramatically decrease actin polymerization induced by MyoGEF. In addition, MyoGEF colocalizes with nonmuscle myosin IIA (NMIIA) to the front of migrating cells, and depletion of NMIIA by RNAi disrupts the polarized localization of MyoGEF at the cell leading edge, suggesting a role for NMIIA in regulating MyoGEF localization and function. Moreover, MyoGEF protein levels significantly increase in infiltrating ductal carcinomas as well as in invasive breast cancer cell lines. Taken together, our results suggest that MyoGEF cooperates with NMIIA to regulate the polarity and invasion activity of breast cancer cells through activation of RhoA and RhoC.
Despite significant progress in the treatment of breast cancer, particularly through the use of t... more Despite significant progress in the treatment of breast cancer, particularly through the use of targeted therapy, relapse and chemoresistance remain a major hindrance to the fight to minimize the burden of the disease. It is becoming increasingly clear that a rare subpopulation of cells known as cancer stem cells (CSC), able to be generated through epithelial-to-mesenchymal transition (EMT) and capable of tumor initiation and self-renewal, contributes to treatment resistance and metastases. This means that a more effective therapy should target both the chemoresistant CSCs and the proliferating epithelial cells that give rise to them to reverse EMT and to attenuate their conversion to CSCs. Here, we demonstrate a novel function of AXL in acting upstream to induce EMT in normal and immortalized human mammary epithelial cells in an apparent positive feedback loop mechanism and regulate breast CSC (BCSC) self-renewal and chemoresistance. Downregulation of AXL using MP470 (Amuvatinib) reversed EMT in mesenchymal normal human mammary epithelial cells and murine BCSCs attenuating self-renewal and restored chemosensitivity of the BCSCs. AXL expression was also found to be associated with the expression of stem cell genes, regulation of metastases genes, increased tumorigenicity and was important for BCSC invasion and migration. Inactivation of AXL also led to the downregulation of nuclear factor-kB pathway and reduced tumor formation in vivo. Taken together, our data suggest that targeted therapy against AXL, in combination with systemic therapies, has the potential to improve response to anticancer therapies and to reduce breast cancer recurrence and metastases.
Cooperative communications between the central spindle and the contractile ring are critical for ... more Cooperative communications between the central spindle and the contractile ring are critical for the spatial and temporal regulation of cytokinesis. Here we report that MyoGEF, a guanine nucleotide exchange factor that localizes to the central spindle and cleavage furrow, interacts with centrosome/spindle pole-associated protein (CSPP), which is concentrated at the spindle pole and central spindle during mitosis and cytokinesis. Both in vitro and in vivo pulldown assays show that MyoGEF interacts with CSPP. The C-terminus of MyoGEF and N-terminus of CSPP are required for their interaction. Immunofluorescence analysis indicates that MyoGEF and CSPP colocalize at the central spindle. Depletion of CSPP or MyoGEF by RNA-interference (RNAi) not only causes defects in mitosis and cytokinesis, such as metaphase arrest and furrow regression, but also mislocalization of nonmuscle myosin II with a phosphorylated myosin regulatory light chain (p-MRLC). Importantly, CSPP depletion by RNAi inter...
We reported previously that a guanine nucleotide exchange factor, MyoGEF, localizes to the centra... more We reported previously that a guanine nucleotide exchange factor, MyoGEF, localizes to the central spindle, activates RhoA, and is required for cytokinesis. In this study, we have found that Plk1 (polo-like kinase 1) can phosphorylate MyoGEF, thereby recruiting MyoGEF to the central spindle as well as enhancing MyoGEF activity toward RhoA. The in vitro kinase assay shows that Plk1 can phosphorylate MyoGEF on threonine 574. Immunoprecipitation/immunoblot analysis demonstrates that mutation of threonine 574 to alanine dramatically decreases threonine phosphorylation of MyoGEF in transfected HeLa cells, suggesting that threonine 574 is phosphorylated in vivo. Consistent with these observations, immunofluorescence shows that Plk1 and MyoGEF colocalize at the spindle pole and central spindle during mitosis and cytokinesis. Importantly, RNA interference-mediated depletion of Plk1 interferes with the localization of MyoGEF at the spindle pole and central spindle. Moreover, mutation of threonine 574 to alanine in MyoGEF or depletion of Plk1 by RNA interference leads to a decrease in MyoGEF activity toward RhoA in HeLa cells. Therefore, our results suggest that Plk1 can regulate MyoGEF activity and localization, contributing to the regulation of cytokinesis.
Background: Aurora B kinase (aurora B) and polo-like kinase 1 (Plk1) are critical regulators of c... more Background: Aurora B kinase (aurora B) and polo-like kinase 1 (Plk1) are critical regulators of cytokinesis. Results: Phosphorylation of MyoGEF at Thr-544 by aurora B promotes the binding of Plk1 to MyoGEF. Conclusion: Phosphorylation of MyoGEF by aurora B creates a docking site for Plk1. Significance: Coordinated regulation of MyoGEF localization and activation by aurora B and Plk1 contributes to the regulation of cytokinesis. We previously reported that phosphorylation of myosin IIinteracting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.
wuCC5-11-vidS1.mov. Localization of GFPtagged MyoGEF in HeLa cells during cytokinesis. HeLa cells... more wuCC5-11-vidS1.mov. Localization of GFPtagged MyoGEF in HeLa cells during cytokinesis. HeLa cells were transfected with a plasmid encoding GFP-tagged MyoGEF by using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the live HeLa cells expressing GFP-tagged MyoGEF were imaged by using a fluorescence Olympus X170 microscope equipped with a cooled CCD camera.
Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess... more Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial–mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of m...
The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single... more The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell–dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24−/loCD44+ phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have seve...
This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (My... more This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (MyoGEF) and centrosome/spindle pole associated protein (CSPP) in mitotic progression and cytokinesis. We have identified three mouse isoforms of CSPP, all of which interact and colocalize with MyoGEF to the central spindle in anaphase cells. The N-terminus of MyoGEF interacts with myosin whereas the C terminus interacts with the N-terminus of CSPP, forming a complex. The N-terminus of CSPP appears to be important for both localization and interaction with MyoGEF. CSPP plays a role in mitotic progression since its depletion by RNAi resulted in metaphase arrest. MyoGEF is required for completion of cytokinesis. Both MyoGEF and CSPP are phosphorylated by mitotic kinases including Plk1 and Aurora. Importantly, MyoGEF is phosphorylated at Thr-574 in mitosis by Polo-like kinase 1, and this phosphorylation is required for activation of RhoA. Thr-543 of MyoGEF is required for Plk1 binding in mitosis and phosphorylation of MyoGEF by Cdk1/cyclinB, possibly at Thr-543 may generate a Plk1 docking site, i.e., Cdk1 can phosphorylate MyoGEF at Thr-543, thereby allowing Plk1 to bind and phosphorylate MyoGEF at Thr-574. Finally, MyoGEF and CSPP are also phosphorylated by Aurora-B kinase in vitro. Taken together, we propose that Aurora-B may phosphorylate and recruit MyoGEF and CSPP to the central spindle, where phosphorylation of MyoGEF at Thr-543 promotes Polo kinase binding and additional phosphorylation of MyoGEF, leading to the activation of RhoA at the cleavage furrow.
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Papers by Michael Asiedu