Kelly Hall

Inflammation induces lymphangiogenesis through up-regulation of VEGFR-3 mediated by NF-κB and Prox1

The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-kappaB) family; however, the role of NF-kappaB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-kappaB pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-kappaB followed by sequential up-regulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-kappaB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells, resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-kappaB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-kappaB pathway by inflammatory stimuli activates Prox1, and both NF-kappaB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of preexisting lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

Lymphangiogenesis and Lymphatic Metastasis in Breast Cancer

Lymphatic metastasis is the main prognostic factor for survival of patients with breast cancer and other epithelial malignancies. Mounting clinical and experimental data suggest that migration of tumor cells into the lymph nodes is greatly facilitated by lymphangiogenesis, a process that generates new lymphatic vessels from pre-existing lymphatics with the aid of circulating lymphatic endothelial progenitor cells. The key protein that induces lymphangiogenesis is vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by vascular endothelial growth factor-C and -D (VEGF-C and VEGF-D). These lymphangiogenic factors are commonly expressed in malignant, tumor-infiltrating and stromal cells, creating a favorable environment for generation of new lymphatic vessels. Clinical evidence demonstrates that increased lymphatic vessel density in and around tumors is associated with lymphatic metastasis and reduced patient survival. Recent evidence shows that breast cancers induce remodeling of the local lymphatic vessels and the regional lymphatic network in the sentinel and distal lymph nodes. These changes include an increase in number and diameter of tumor-draining lymphatic vessels. Consequently, lymph flow away from the tumor is increased, which significantly increases tumor cell metastasis to draining lymph nodes and may contribute to systemic spread. Collectively, recent advances in the biology of tumor-induced lymphangiogenesis suggest that chemical inhibitors of this process may be an attractive target for inhibiting tumor metastasis and cancer-related death. Nevertheless, this is a relatively new field of study and much remains to be established before the concept of tumor-induced lymphangiogenesis is accepted as a viable anti-metastatic target. This review summarizes the current concepts related to breast cancer lymphangiogenesis and lymphatic metastasis while highlighting controversies and unanswered questions.

Paclitaxel Therapy Promotes Breast Cancer Metastasis in a TLR4-Dependent Manner

Emerging evidence suggests that cytotoxic therapy may actually promote drug resistance and metastasis while inhibiting the growth of primary tumors. Work in preclinical models of breast cancer has shown that acquired chemoresistance to the widely used drug paclitaxel can be mediated by activation of the Toll-like receptor TLR4 in cancer cells. In this study, we determined the prometastatic effects of tumor-expressed TLR4 and paclitaxel therapy and investigated the mechanisms mediating these effects. While paclitaxel treatment was largely efficacious in inhibiting TLR4-negative tumors, it significantly increased the incidence and burden of pulmonary and lymphatic metastasis by TLR4-positive tumors. TLR4 activation by paclitaxel strongly increased the expression of inflammatory mediators, not only locally in the primary tumor microenvironment but also systemically in the blood, lymph nodes, spleen, bone marrow, and lungs. These proinflammatory changes promoted the outgrowth of Ly6C(+) and Ly6G(+) myeloid progenitor cells and their mobilization to tumors, where they increased blood vessel formation but not invasion of these vessels. In contrast, paclitaxel-mediated activation of TLR4-positive tumors induced de novo generation of deep intratumoral lymphatic vessels that were highly permissive to invasion by malignant cells. These results suggest that paclitaxel therapy of patients with TLR4-expressing tumors may activate systemic inflammatory circuits that promote angiogenesis, lymphangiogenesis, and metastasis, both at local sites and premetastatic niches where invasion occurs in distal organs. Taken together, our findings suggest that efforts to target TLR4 on tumor cells may simultaneously quell local and systemic inflammatory pathways that promote malignant progression, with implications for how to prevent tumor recurrence and the establishment of metastatic lesions, either during chemotherapy or after it is completed.

Regulation of tumor angiogenesis by the local environment.

Angiogenesis is the process of formation of new blood vessels from pre-existing vessels or endothelial cell progenitors. It plays an essential role in embryogenesis, inflammation, wound healing, tumor growth and metastasis. The tumor microenvironment contains excessive amounts of pro-angiogenic factors derived from neoplastic, stromal, and infiltrating immune cells. The imbalance of pro-angiogenic and anti-angiogenic factors promotes abnormal angiogenesis, creating numerous blood vessels with structural abnormalities and functional defects. These defective vessels often create an inflammatory environment within the tumor that promotes coagulation, thrombosis, and impairs blood supply, causing further complications to the cancer patient. The structural and functional abnormalities of the tumor vessels promote hematogenous metastasis, which is strongly associated with shorter patient survival. Furthermore, tumor blood vessels are poorly perfused, which impedes drug delivery to the tumor, thus reducing the efficacy of anti-cancer agents. Tumor angiogenesis is widely studied as an important target for suppressing tumor growth and metastasis. This review will briefly summarize the current findings related to regulation of angiogenesis by the tumor microenvironment, while highlighting potential targets for inhibiting this process.

EXPRESSION OF A NOVEL MARKER, Ubc9, IN SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK

Ubiquitin‐conjugating enzyme (Ubc9) is a novel enzyme involved in posttranslational modification of cellular proteins. The objective of this study was to determine the expression of Ubc9 in squamous cell carcinoma of the head and neck (SCCHN).

IRF5 is a novel regulator of CXCL13 expression in breast cancer that regulates CXCR5(+) B- and T-cell trafficking to tumor-conditioned media.

Clinical studies using prognostic and predictive signatures have shown that an immune signal emanating from whole tumors reflects the level of immune cell infiltration—a high immune signal linked to improved outcome. Factors regulating immune cell trafficking to the tumor, however, are not known. Previous work has shown that expression of interferon regulatory factor 5 (IRF5), a critical immune regulator, is lost in ~80% of invasive ductal carcinomas examined. We postulated that IRF5‐positive and ‐negative breast tumors would differentially regulate immune cell trafficking to the tumor. Using a focused tumor inflammatory array, differences in cytokine and chemokine expression were examined between IRF5‐positive and ‐negative MDA‐MB‐231 cells grown in three‐dimensional culture. A number of cytokines/chemokines were found to be dysregulated between cultures. CXCL13 was identified as a direct target of IRF5 resulting in the enhanced recruitment of B and T cells to IRF5‐positive tumor‐conditioned media. The ability of IRF5 to regulate mediators of cell migration was confirmed by enzyme‐linked immunosorbent assay, chromatin immunoprecipitation assay, small interfering RNA knockdown and immunofluorescence staining of human breast tumor tissues. Analysis of primary immune cell subsets revealed that IRF5 specifically recruits CXCR5+ B and T cells to the tumor; CXCR5 is the receptor for CXCL13. Analysis of primary breast tumor tissues revealed a significant correlation between IRF5 and CXCL13 expression providing clinical relevance to the study. Together, these data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may, in combination with CXCL13, serve as a novel prognostic marker for antitumor immunity.

Lymphatic endothelial progenitors originate from plastic myeloid cells activated by toll-like receptor-4

Background Myeloid-derived lymphatic endothelial cells (M-LECP) are induced by inflammation and play an important role in adult lymphangiogenesis. However, the mechanisms driving M-LECP differentiation are currently unclear. We previously showed that activation of Toll-like receptor-4 (TLR4) induces myeloid-lymphatic transition (MLT) of immortalized mouse myeloid cells. Here the goals were to assess the potential of different TLR4 ligands to induce pro-lymphatic reprogramming in human and mouse primary myeloid cells and to identify transcriptional changes regulating this process. Methodology/Principal findings Human and mouse myeloid cells were reprogrammed to the lymphatic phenotype by TLR4 ligands including lipopolysaccharide (LPS), recombinant high mobility group box 1 protein (HMGB1), and paclitaxel. TLR4 induced similar MLT in cells from mice of different strains and immune status. Commonly induced genes were detected by transcriptional profiling in human and mouse myeloid cells from either immunocompetent or immunodeficient mice. Shared trends included: (1) novel expression of lymphatic-specific markers vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and podoplanin (PDPN) largely absent prior to induction; (2) lack of notable changes in blood vessel-specific markers; (3) transient expression of VEGFR-3, but sustained increase of vascular endothelial growth factor-C (VEGF-C) and a variety of inflammatory cytokines; (4) dependency of VEGFR-3 upregulation and other LEC genes on NF-κB; and (5) novel expression of lymphatic-specific (e.g., PROX1) and stem/progenitor (e.g., E2F1) transcription factors known for their roles in adult and embryonic vascular formation. M-LECP generated by TLR4 ligands in vitro were functional in vivo as demonstrated by significantly increased lymphatic vessel density and lymphatic metastasis detected in orthotopic breast cancer models. Conclusions/Significance We established a novel TLR4-dependent protocol for in vitro production of functionally competent M-LECP from primary human or mouse myeloid cells and identified many potential regulators of this process. This information can be further exploited for research and therapeutic purposes.

Abstract 1460: Bone marrow-derived CD11b+/Podoplanin+ cells are lymphatic progenitors directly responsible for breast cancer lymphatic formation

Introduction: Lymphatic metastasis, a key factor for poor outcome of breast cancer (BC), strongly depends on lymphatic vessel (LV) formation. Recent studies suggest that lymphangiogenesis is strongly promoted by bone marrow (BM)-derived CD11b + monocytes that differentiate into Lymphatic Endothelial Cell Progenitors dubbed here M-LECP. While BC are known to recruit BM monocytes, a specific BM subset responsible for tumor lymphatic formation has not been identified. We recently discovered that podoplanin (Pdpn) is the highest upregulated lymphatic marker that signifies transdifferentiation of either human or mouse immature myeloid cells into M-LECP. We hypothesized that BM-derived M-LECP are represented by a subset expressing podoplanin in CD11b + myeloid cells that are capable of inducing tumor lymphatics. Methods: Tumor lymphatic vessels expressing myeloid markers were detected immunohistochemically by triple-staining in clinical specimens and experimental breast tumors. Expression of Vegfr-3, Lyve-1, Sca-1, and other markers in BM-derived CD11b + /Pdpn + and Pdpn-negative subsets was determined by FACS. Expression of lymphatic-specific markers in CD11b + /Pdpn + and Pdpn − myeloid cells isolated from MDA-MB-231 tumors was determined by RT-qPCR. CD11b + /Pdpn + and Pdpn-negative subsets from BM of GFP-expressing mice were adoptively transferred to mice with orthotopic breast tumors followed by quantifying mobilized GFP + /CD11b + /Pdpn + cells and the density of tumor LV. Results: M-LECP were absent in normal human breast tissues but highly present in tumors. Analysis of clinical BC specimens showed significant correlations between tumor-mobilized M-LECP, density of LV and metastasis to regional nodes. All examined BC models exhibited very high densities (60-90%) of double-positive macrophages expressing lymphatic markers, and lymphatic vessels expressing myeloid proteins. At least a half of CD11b + cells isolated from tumors were positive for podoplanin and nearly 90% of CD11b + /Pdpn + subset expressed markers of progenitor cells. Only CD11b + /Pdpn + subset (but not other myeloid cells) expressed a broad panel of proteins specific to the lymphatic endothelial lineage. Adoptive transfer of BM-derived CD11b + /Pdpn + or Pdpn-negative fractions into tumor-bearing mice showed that only Pdpn+ BM myeloid cells integrated into preexisting LV and caused a 10-fold increase in peritumoral and intratumoral LV density. Conclusion: We show for the first time in clinical BC samples that tumor-recruited M-LECP significantly correlate with LN metastasis. We also identified a phenotypically distinct BM fraction of CD11b + /Pdpn + cells that directly promote generation of new tumor lymphatic vessels. Our data suggest that tumors induce myeloid BM cells to undergo differentiation into M-LECP that subsequently promote cancer lymphatics thereby promoting tumor spread. Citation Format: Caitlin Griggs, Kelly Hall, Lisa Volk-Draper, Kathy Robinson, Sophia Ran. Bone marrow-derived CD11b + /Podoplanin + cells are lymphatic progenitors directly responsible for breast cancer lymphatic formation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1460.

Abstract 771: TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis

Background: Paclitaxel (PXL) is a widely-used drug for breast cancer therapy. Resistance, however, occurs frequently and the evasion mechanisms remain unclear. PXL is known to activate Toll-like Receptor-4 (TLR4), a major signaling receptor expressed on immune cells responding to lipopolysaccharide (LPS). LPS activation of TLR4 in the immune cells substantially enhances their migratory, invasive, proliferative, and pro-survival functions as necessary for the body defense. Since PXL is a functional LPS mimetic, we hypothesized that acquisition of such phenotype by TLR4-expressing cancer cells promotes tumor growth, metastasis, and resistance to therapy. Methods: We tested this hypothesis in two breast cancer luciferase-tagged models genetically modified to either suppress TLR4 expression in a positive line, MDA-MB-231, or overexpress it in a negative line, HCC1806. Modified cell lines and their controls were orthotopically implanted in mice followed by measuring tumor growth prior and post-PXL treatment. Incidence and burden of lymph node (LN) and lung metastases were quantified by measuring luciferase activity in respective organs. TLR4-induced inflammation was assessed by measuring cytokines in blood, tumor lysates, and metastatic organs using RT-qPCR and ELISA. Cell composition of spleens and bone marrow from control and treated mice were analyzed by FACS. TLR4-induced changes in tumor vasculature were determined by immunostaining for blood and lymphatic vessel markers. Results: TLR4 expressed in tumor cells significantly increased rate of recurrence and metastasis, an event augmented by PXL treatment. Local inflammation was also enhanced by the PXL•TLR4 axis as illustrated by a 276-fold increase in IL-6 in lysates of TLR4+ tumors from treated mice as compared with samples of isogenic tumors with depleted TLR4. Activation of this pathway also increased cytokine levels in the blood and distant organs as indicted by 3-6 fold increase in IL-4 and IL-10 in LNs and lungs. These pro-inflammatory systemic changes promoted generation of myeloid progenitors in bone marrow and spleen evident by 4-5 fold increase in Ly6C+ cell population in TLR4-overexpressing tumors compared with those lacking TLR4. Importantly, activation of the PXL•TLR4 axis induced intratumoral formation of lymphatic vessels in HCC1806 tumors absent in all other experimental groups. Conclusion: These data imply that PXL therapy activates TLR4 often overexpressed in breast cancer. Activation of this pathway substantially increases local tumor and systemic inflammation leading to generation of myeloid progenitors that can promote metastasis by inducing angiogenesis and lymphatic vessel formation, particularly inside the tumor. These findings suggest that tumor expression of TLR4 may indicate poor prognosis and response to therapy, and that blocking the TLR4 pathway may improve current anti-cancer treatments. Citation Format: Sandeep Rajput, Lisa Volk-Draper, Kelly Hall, Sophia Ran. TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2014-771

Abstract 12: Tumor macrophages in clinical breast cancers transdifferentiate into lymphatic-like cells and structurally contribute to lymphatic vasculature

Background: Lymph node (LN) metastasis, the strongest prognostic factor for breast cancer (BC) patient survival, directly correlates with increased density of lymphatic vessels. The formation of new lymphatic vessels (i.e. lymphangiogenesis) is strongly promoted by tumor-associated macrophages (TAMs) through mechanisms that are not fully understood. We recently discovered that inflammatory lymphangiogenesis is driven by bone marrow derived myeloid cells that transdifferentiate into lymphatic-like cells and structurally contribute to growing lymphatic vasculature. Clinical BC are characterized by an inflammatory environment and massive recruitment of macrophages. We, therefore, hypothesize that BC mobilized monocytic precursors may promote tumor lymphangiogenesis by induction of their transdifferentiation into Macrophage-derived Lymphatic Endothelial Cell Progenitors designated here as M-LECPs. Methodology: To test this hypothesis, we analyzed 75 clinical specimens of invasive breast carcinoma and 5 normal breast tissues for the presence of M-LECPs and incidence of their integration into lymphatic vessels. M-LECPs were identified by double or triple staining using multiple specific markers for myeloid (CD68, CD14, and CD11b) and lymphatic lineages (LYVE-1, podoplanin and VEGFR-3). Integrated M-LECPs were identified by double staining of LYVE-1 + vascular structures with antibodies against myeloid-specific markers as well as confocal microscopy using anti-VE-cadherin staining. Results: Out of 75 tumors, 58% had lymphatics, and 46% of these specimens showed integration of M-LECPs in the lymphatic vessels. In stark contrast, normal mammary tissues showed less than 5% of either double-stained macrophages or dual-identity vessels. The predominant M-LECP association with tumors was also evident in metastatic BC mouse models including MDA-MB-231 (human xenograft), R3L (transplantable syngeneic), and MMTV-PyMT (spontaneous syngeneic). All three models contained 40-50% of TAMs positive for lymphatic markers and up to 90% of lymphatic vessels positive for myeloid markers. In vitro studies showed that inflammatory triggers induce lymphatic proteins in myeloid cells but not myeloid markers in inflamed lymphatic endothelial cells. Conclusions/Significance: These findings show, for the first time, that majority of TAMs in clinical BC exhibit the lymphatic phenotype and integrate into lymphatic vessels. Reproduction of this phenomenon in three BC mouse models and cultured myeloid cells treated with inflammatory mediators suggests a strong self-autonomous role of TAMs into outgrowth of tumor lymphatics. Further investigation of this novel mechanism of TAM-dependent lymphangiogenesis may identify new targets for inhibiting lymphatic metastasis leading to improved survival of breast cancer patients. Citation Format: Kelly Hall, Lisa Volk-Draper, Sandeep Rajput, David DeNardo, Sophia Ran. Tumor macrophages in clinical breast cancers transdifferentiate into lymphatic-like cells and structurally contribute to lymphatic vasculature. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 12. doi:10.1158/1538-7445.AM2014-12