Gaofeng Xiong

Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition.

BackgroundIncreased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated.MethodsGene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis.ResultsUsing gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models.ConclusionThese results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression.

Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology

MicroRNAs play important roles in regulating the gene expression and life cycle of cancer cells. In particular, miR-21, an oncogenic miRNA is a major player involved in tumor initiation, progression, invasion and metastasis in several cancers, including triple negative breast cancer (TNBC). However, delivery of therapeutic miRNA or anti-miRNA specifically into cancer cells in vivo without collateral damage to healthy cells remains challenging. We report here the application of RNA nanotechnology for specific and efficient delivery of anti-miR-21 to block the growth of TNBC in orthotopic mouse models. The 15 nm therapeutic RNA nanoparticles contains the 58-nucleotide (nt) phi29 pRNA-3WJ as a core, a 8-nt sequence complementary to the seed region of miR-21, and a 39-nt epidermal growth factor receptor (EGFR) targeting aptamer for internalizing RNA nanoparticles into cancer cells via receptor mediated endocytosis. The RNase resistant and thermodynamically stable RNA nanoparticles remained intact after systemic injection into mice and strongly bound to tumors with little or no accumulation in healthy organs 8 h postinjection, and subsequently repressed tumor growth at low doses. The observed specific cancer targeting and tumor regression is a result of several key attributes of RNA nanoparticles: anionic charge which disallows nonspecific passage across negatively charged cell membrane; “active” targeting using RNA aptamers which increases the homing of RNA nanoparticles to cancer cells; nanoscale size and shape which avoids rapid renal clearance and engulfment by lung macrophages and liver Kupffer cells; favorable biodistribution profiles with little accumulation in healthy organs, which minimizes nonspecific side effects; and favorable pharmacokinetic profiles with extended in vivo half-life. The results demonstrate the clinical potentials of RNA nanotechnology based platform to deliver miRNA based therapeutics for cancer treatment.

RORα Suppresses Breast Tumor Invasion by Inducing SEMA3F Expression

Inactivation of tumor suppressors and inhibitory microenvironmental factors is necessary for breast cancer invasion; therefore, identifying those suppressors and factors is crucial not only to advancing our knowledge of breast cancer, but also to discovering potential therapeutic targets. By analyzing gene expression profiles of polarized and disorganized human mammary epithelial cells in a physiologically relevant three-dimensional (3D) culture system, we identified retinoid orphan nuclear receptor alpha (RORα) as a transcription regulator of semaphorin 3F (SEMA3F), a suppressive microenvironmental factor. We showed that expression of RORα was downregulated in human breast cancer tissue and cell lines, and that reduced mRNA levels of RORα and SEMA3F correlated with poor prognosis. Restoring RORα expression reprogrammed breast cancer cells to form noninvasiveness structures in 3D culture and inhibited tumor growth in nude mice, accompanied by enhanced SEMA3F expression. Inactivation of RORα in nonmalignant human mammary epithelial cells inhibited SEMA3F transcription and impaired polarized acinar morphogenesis. Using chromatin immunoprecipitation and luciferase reporter assays, we showed that transcription of SEMA3F is directly regulated by RORα. Knockdown of SEMA3F in RORα-expressing cancer cells rescued the aggressive 3D phenotypes and tumor invasion. These findings indicate that RORα is a potential tumor suppressor and inhibits tumor invasion by inducing suppressive cell microenvironment.

Chaperone Hsp47 drives malignant growth and invasion by modulating an ECM gene network

The extracellular matrix (ECM) is a determining factor in the tumor microenvironment that restrains or promotes malignant growth. In this report, we show how the molecular chaperone protein Hsp47 functions as a nodal hub in regulating an ECM gene transcription network. A transcription network analysis showed that Hsp47 expression was activated during breast cancer development and progression. Hsp47 silencing reprogrammed human breast cancer cells to form growth-arrested and/or noninvasive structures in 3D cultures, and to limit tumor growth in xenograft assays by reducing deposition of collagen and fibronectin. Coexpression network analysis also showed that levels of microRNA(miR)-29b and -29c were inversely correlated with expression of Hsp47 and ECM network genes in human breast cancer tissues. We found that miR-29 repressed expression of Hsp47 along with multiple ECM network genes. Ectopic expression of miR-29b suppressed malignant phenotypes of breast cancer cells in 3D culture. Clinically, increased expression of Hsp47 and reduced levels of miR-29b and -29c were associated with poor survival outcomes in breast cancer patients. Our results show that Hsp47 is regulated by miR-29 during breast cancer development and progression, and that increased Hsp47 expression promotes cancer progression in part by enhancing deposition of ECM proteins.

Integrated extracellular matrix signaling in mammary gland development and breast cancer progression.

Extracellular matrix (ECM), a major component of the cellular microenvironment, plays critical roles in normal tissue morphogenesis and disease progression. Binding of ECM to membrane receptor proteins, such as integrin, discoidin domain receptors, and dystroglycan, elicits biochemical and biomechanical signals that control cellular architecture and gene expression. These ECM signals cooperate with growth factors and hormones to regulate cell migration, differentiation, and transformation. ECM signaling is tightly regulated during normal mammary gland development. Deposition and alignment of fibrillar collagens direct migration and invasion of mammary epithelial cells during branching morphogenesis. Basement membrane proteins are required for polarized acinar morphogenesis and milk protein expression. Deregulation of ECM proteins in the long run is sufficient to promote breast cancer development and progression. Recent studies demonstrate that the integrated biophysical and biochemical signals from ECM and soluble factors are crucial for normal mammary gland development as well as breast cancer progression.

Function of cancer cell-derived extracellular matrix in tumor progression

Extracellular matrix (ECM) is an essential component of the tumor microenvironment. Cancer development and progression are associated with increased ECM deposition and crosslink. The chemical and physical signals elicited from ECM are necessary for cancer cell proliferation and invasion. It is well recognized that stromal cells are a major source of ECM proteins. However, recent studies showed that cancer cells are also an active and important component in ECM remodeling. Cancer cells deposit a significant amount of collagen, fibronectin, and tenascin C (TNC). Recent studies demonstrate that these cancer cell-derived ECM proteins enhance cancer cell survival and promote cancer cell colonization at distant sites. ECM-related enzymes and chaperone proteins, such as prolyl-4-hydroxylase, lysyl-hydroxylase, lysyl oxidase, and heat shock protein 47, are also highly expressed in cancer cells. Inhibition of these enzymes significantly reduces cancer growth, invasion, and metastasis. These factors suggest that the cancer cell-derived ECM is crucial for cancer progression and metastasis. Therefore, targeting these ECM proteins and ECM-related enzymes is a potential strategy for cancer treatment.

Abstract 2701: RORα suppresses breast tumor progression by inducing cell polarization and inhibiting cell invasion

Background: Disruption of tissue polarity is one of hallmarks of breast cancer development, which is associated with cell invasion and cancer dissemination. Therefore, identifying proteins and pathways that modulate cell polarity is crucial not only to advancing our knowledge of breast cancer, but also to discovering potential therapeutic targets. Methods: To identify potential regulators of tissue polarity, we analyzed over-represented DNA motifs in the promoter regions of the polarization genes that are significantly up-regulated genes in polarized human mammary epithelial cells in a physiologically relevant three-dimensional (3D) culture system. We assessed the protein levels of RORα by immunoblotting and immunohistochemistry in malignant and non-malignant human mammary epithelial cells as well as human breast cancer tissue array. To explore the functional significance of RORα down-regulation the malignance of breast cancer cells, we restored RORα expression in malignant T4-2 cells, MDA231 cells and silenced RORα expression in non-malignant S1 cells. The growth and malignant phenotypes of these cells were examined in 3D culture and xenograft model. Results: Results from promoter analysis suggest that RORα is a potential regulator of mammary tissue polarity and is inactivated in disorganized HMECs. Expression of RORα protein is significantly reduced in malignant breast cancer cell lines and this reduction is associated with disruption of acinar structure in 3D culture. Both mRNA and protein level of RORα were significantly reduced in human breast cancer tissues. Restoring RORα expression in T4-2 and MDA-MB 231 cells significantly suppressed proliferation, invasion and the malignant phenotypes in 3D culture. Silencing RORα expression in non-malignant S1 cells disrupts the polarized acinar morphogenesis. Restoring RORα expression in T4-2 and MDA-MB 231 cells also inhibits the tumor growth in nude mice. Moreover, patients with reduced expression of RORα in cancer tissue have significantly shorter survival. Conclusion: Together these results indicate that inactivation of RORα promotes the breast cancer development and progression by enhancing cell invasion and disturbing normal tissue architecture. RORα provide a potential therapeutic target for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2701. doi:1538-7445.AM2012-2701

Increased ROS production in non-polarized mammary epithelial cells induces monocyte infiltration in 3D culture

ABSTRACT Loss of epithelial cell polarity promotes cell invasion and cancer dissemination. Therefore, identification of factors that disrupt polarized acinar formation is crucial. Reactive oxygen species (ROS) drive cancer progression and promote inflammation. Here, we show that the non-polarized breast cancer cell line T4-2 generates significantly higher ROS levels than polarized S1 and T4R cells in three-dimensional (3D) culture, accompanied by induction of the nuclear factor κB (NF-κB) pathway and cytokine expression. Minimizing ROS in T4-2 cells with antioxidants reestablished basal polarity and inhibited cell proliferation. Introducing constitutively activated RAC1 disrupted cell polarity and increased ROS levels, indicating that RAC1 is a crucial regulator that links cell polarity and ROS generation. We also linked monocyte infiltration with disruption of polarized acinar structure using a 3D co-culture system. Gain- and loss-of-function experiments demonstrated that increased ROS in non-polarized cells is necessary and sufficient to enhance monocyte recruitment. ROS also induced cytokine expression and NF-κB activity. These results suggest that increased ROS production in mammary epithelial cell leads to disruption of cell polarity and promotes monocyte infiltration. Summary: Increased ROS levels contribute to the disruption of polarized acinar structures and induce monocyte recruitment through induction of the NF-κB pathway and cytokine expression.

RORα Binds to E2F1 To Inhibit Cell Proliferation and Regulate Mammary Gland Branching Morphogenesis

ABSTRACT Retinoic acid receptor-related orphan nuclear receptor alpha (RORα) is a potent tumor suppressor that reduces cell proliferation and inhibits tumor growth. However, the molecular mechanism by which it inhibits cell proliferation remains unknown. We demonstrate a noncanonical nuclear receptor pathway in which RORα binds to E2F1 to inhibit cell cycle progression. We showed that RORα bound to the heptad repeat and marked box region of E2F1 and suppressed E2F1-regulated transcription in epithelial cells. Binding of RORα inhibited E2F1 acetylation and its DNA-binding activity by recruiting histone deacetylase 1 (HDAC1) to the protein complexes. Knockdown of HDAC1 or inhibition of HDAC activity at least partially rescued transcription factor activity of E2F1 that was repressed by RORα. Importantly, RORα levels were increased in mammary ducts compared to terminal end buds and inversely correlated with expression of E2F1 target genes and cell proliferation. Silencing RORα in mammary epithelial cells significantly enhanced cell proliferation in the ductal epithelial cells and promoted side branching of the mammary ducts. These results reveal a novel link between RORα and E2F1 in regulating cell cycle progression and mammary tissue morphogenesis.

Abstract 2374: Mir-29/Hsp47 regulate breast cancer progression by modulating the ECM transcription network

The extracellular matrix (ECM), an essential component of tumor microenvironment, play crucial roles in cancer development and progression. Using gene co-expression network analysis, we have identified an ECM transcription network from hundreds of human breast cancer. In this report, we show how the molecular chaperone protein Hsp47 functions as a nodal hub in regulating an ECM gene transcription network. We found that Hsp47 expression was activated during breast cancer development and progression. Hsp47 silencing reprogrammed human breast cancer cells to form growth-arrested and/or non-invasive structures in 3D cultures, and to limit tumor growth in xenograft assays by reducing deposition of collagen and fibronectin. Co-expression network analysis also showed that levels of microRNA-29b and 29c were inversely correlated with expression of Hsp47 and ECM network genes in human breast cancer tissues. We found that miR-29 repressed expression of Hsp47 along with multiple ECM network genes. Ectopic expression of miR-29b suppressed malignant phenotypes of breast cancer cells in 3D culture. Clinically, increased expression of Hsp47 and reduced levels of miR-29b and 29c were associated with poor survival outcomes in breast cancer patients. Our results show that Hsp47 is regulated by miR-29 during breast cancer development and progression, and that increased Hsp47 expression promotes cancer progression in part by enhancing deposition of ECM proteins. Citation Format: Jieqing Zhu, Gaofeng Xiong, Ren Xu. Mir-29/Hsp47 regulate breast cancer progression by modulating the ECM transcription network. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2374. doi:10.1158/1538-7445.AM2015-2374