Dongwon Choi

Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis

Background:Colorectal cancer (CRC) is a leading cause of death in the United States. Increased level of interleukin-8 (IL-8) and CXCR2 on tumours and in the tumour microenvironment has been associated with CRC growth, progression and recurrence in patients. Here, we aimed to evaluate the effects of tissue microenvironment-encoded IL-8 and CXCR2 on colon cancer progression and metastasis.Methods:A novel immunodeficient, skin-specific IL-8-expressing transgenic model was generated to evaluate colon cancer growth and metastasis. Syngeneic mouse colon cancer cells were grafted in CXCR2 knockout (KO) mice to study the contribution of CXCR2 in the microenvironment to cancer growth.Results:Elevated levels of IL-8 in the serum and tumour microenvironment profoundly enhanced the growth of human and mouse colon cancer cells with increased peri-tumoural angiogenesis, and also promoted the extravasation of the cancer cells into the lung and liver. The tumour growth was inhibited in CXCR2 KO mice with significantly reduced tumour angiogenesis and increased tumour necrosis.Conclusion:Increased expression of IL-8 in the tumour microenvironment enhanced colon cancer growth and metastasis. Moreover, the absence of its receptor CXCR2 in the tumour microenvironment prevented colon cancer cell growth. Together, our study demonstrates the critical roles of the tumour microenvironment-encoded IL-8/CXCR2 in colon cancer pathogenesis, validating the pathway as an important therapeutic target.

Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse

Although the blood vessel-specific fluorescent transgenic mouse has been an excellent tool to study vasculogenesis and angiogenesis, a lymphatic-specific fluorescent mouse model has not been established to date. Here we report a transgenic animal model that expresses the green fluorescent protein under the promoter of Prox1, a master control gene in lymphatic development. Generated using an approximately 200-kb-long bacterial artificial chromosome harboring the entire Prox1 gene, this Prox1-green fluorescent protein mouse was found to faithfully recapitulate the expression pattern of the Prox1 gene in lymphatic endothelial cells and other Prox1-expressing organs, and enabled us to conveniently visualize detailed structure and morphology of lymphatic vessels and networks throughout development. Our data demonstrate that this novel transgenic mouse can be extremely useful for detection, imaging, and isolation of lymphatic vessels and monitoring wound-associated lymphangiogenesis. Together, this Prox1-green fluorescent protein transgenic mouse will be a great tool for the lymphatic research.

Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity

Schlemms canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin α9 and exhibited continuous vascular endothelial-cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.

9-Cis Retinoic Acid Promotes Lymphangiogenesis and Enhances Lymphatic Vessel Regeneration: Therapeutic Implications of 9-Cis Retinoic Acid for Secondary Lymphedema

Background— The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction caused by genetic defects, or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swelling often associated with fibrosis and recurrent infections with no available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema. Methods and Results— We report that RAs promote proliferation, migration, and tube formation of cultured lymphatic endothelial cells by activating fibroblast growth factor receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2, and the aurora kinases through both an Akt-mediated nongenomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals in mouse trachea, Matrigel plug, and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration. Conclusion— These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients. # Clinical Perspective {#article-title-36}Background— The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction caused by genetic defects, or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swelling often associated with fibrosis and recurrent infections with no available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema. Methods and Results— We report that RAs promote proliferation, migration, and tube formation of cultured lymphatic endothelial cells by activating fibroblast growth factor receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2, and the aurora kinases through both an Akt-mediated nongenomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals in mouse trachea, Matrigel plug, and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration. Conclusion— These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients.

Interleukin-8 reduces post-surgical lymphedema formation by promoting lymphatic vessel regeneration

Lymphedema is mainly caused by lymphatic obstruction and manifested as tissue swelling, often in the arms and legs. Lymphedema is one of the most common post-surgical complications in breast cancer patients and presents a painful and disfiguring chronic illness that has few treatment options. Here, we evaluated the therapeutic potential of interleukin (IL)-8 in lymphatic regeneration independent of its pro-inflammatory activity. We found that IL-8 promoted proliferation, tube formation, and migration of lymphatic endothelial cells (LECs) without activating the VEGF signaling. Additionally, IL-8 suppressed the major cell cycle inhibitor CDKN1C/p57KIP2 by downregulating its positive regulator PROX1, which is known as the master regulator of LEC-differentiation. Animal-based studies such as matrigel plug and cornea micropocket assays demonstrated potent efficacy of IL-8 in activating lymphangiogenesis in vivo. Moreover, we have generated a novel transgenic mouse model (K14-hIL8) that expresses human IL-8 in the skin and then crossed with lymphatic-specific fluorescent (Prox1-GFP) mouse. The resulting double transgenic mice showed that a stable expression of IL-8 could promote embryonic lymphangiogenesis. Moreover, an immunodeficient IL-8-expressing mouse line that was established by crossing K14-hIL8 mice with athymic nude mice displayed an enhanced tumor-associated lymphangiogenesis. Finally, when experimental lymphedema was introduced, K14-hIL8 mice showed an improved amelioration of lymphedema with an increased lymphatic regeneration. Together, we report that IL-8 can activate lymphangiogenesis in vitro and in vivo with a therapeutic efficacy in post-surgical lymphedema.

Laminar flow downregulates Notch activity to promote lymphatic sprouting

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow–induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow–induced lymphatic sprouting.

Opposing Regulation of PROX1 by Interleukin-3 Receptor and NOTCH Directs Differential Host Cell Fate Reprogramming by Kaposi Sarcoma Herpes Virus

Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

Lymphatic Reprogramming by Kaposi Sarcoma Herpes Virus Promotes the Oncogenic Activity of the Virus-Encoded G-protein–Coupled Receptor

Kaposi sarcoma, the most common cancer in HIV-positive individuals, is caused by endothelial transformation mediated by the Kaposi sarcoma herpes virus (KSHV)-encoded G-protein-coupled receptor (vGPCR). Infection of blood vascular endothelial cells (BEC) by KSHV reactivates an otherwise silenced embryonic program of lymphatic differentiation. Thus, Kaposi sarcoma tumors express numerous lymphatic endothelial cell (LEC) signature genes. A key unanswered question is how lymphatic reprogramming by the virus promotes tumorigenesis leading to Kaposi sarcoma formation. In this study, we present evidence that this process creates an environment needed to license the oncogenic activity of vGPCR. We found that the G-protein regulator RGS4 is an inhibitor of vGPCR that is expressed in BECs, but not in LECs. RGS4 was downregulated by the master regulator of LEC differentiation PROX1, which is upregulated by KSHV and directs KSHV-induced lymphatic reprogramming. Moreover, we found that KSHV upregulates the nuclear receptor LRH1, which physically interacts with PROX1 and synergizes with it to mediate repression of RGS4 expression. Mechanistic investigations revealed that RGS4 reduced vGPCR-enhanced cell proliferation, migration, VEGF expression, and Akt activation and suppressed tumor formation induced by vGPCR. Our findings resolve long-standing questions about the pathologic impact of KSHV-induced reprogramming of host cell identity, and they offer biologic and mechanistic insights supporting the hypothesis that a lymphatic microenvironment is more favorable for Kaposi sarcoma tumorigenesis.

ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4

Rationale: Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. Objective: We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. Methods and Results: Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress–induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release–activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. Conclusions: Our study identified a molecular mechanism for laminar flow–activated LEC proliferation.

Aberrant activation of Notch signaling inhibits PROX1 activity to enhance the malignant behavior of thyroid cancer cells

Papillary thyroid cancer (PTC) is one of the most common endocrine malignancies associated with significant morbidity and mortality. Although multiple studies have contributed to a better understanding of the genetic alterations underlying this frequently arising disease, the downstream molecular effectors that impact PTC pathogenesis remain to be further defined. Here, we report that the regulator of cell fate specification, PROX1, becomes inactivated in PTC through mRNA downregulation and cytoplasmic mislocalization. Expression studies in clinical specimens revealed that aberrantly activated NOTCH signaling promoted PROX1 downregulation and that cytoplasmic mislocalization significantly altered PROX1 protein stability. Importantly, restoration of PROX1 activity in thyroid carcinoma cells revealed that PROX1 not only enhanced Wnt/β-catenin signaling but also regulated several genes known to be associated with PTC, including thyroid cancer protein (TC)-1, SERPINA1, and FABP4. Furthermore, PROX1 reexpression suppressed the malignant phenotypes of thyroid carcinoma cells, such as proliferation, motility, adhesion, invasion, anchorage-independent growth, and polyploidy. Moreover, animal xenograft studies demonstrated that restoration of PROX1 severely impeded tumor formation and suppressed the invasiveness and the nuclear/cytoplasmic ratio of PTC cells. Taken together, our findings demonstrate that NOTCH-induced PROX1 inactivation significantly promotes the malignant behavior of thyroid carcinoma and suggest that PROX1 reactivation may represent a potential therapeutic strategy to attenuate disease progression.