Ethel R. Pereira

Transcriptional and Post-Transcriptional Regulation of Proangiogenic Factors by the Unfolded Protein Response

Background Inadequate extracellular conditions can adversely affect the environment of the ER and impinge on the maturation of nascent proteins. The resultant accumulation of unfolded proteins activates a signal transduction pathway, known as the unfolded protein response, which serves primarily to protect the cell during stress and helps restore homeostasis to the ER. Principal Findings Microarray analysis of the unfolded protein response in a human medulloblastoma cell line treated with thapsigargin revealed that, in addition to known targets, a large number of proangiogenic factors were up-regulated. Real-Time PCR analyses confirmed that four of these factors, VEGFA, FGF2, angiogenin and IL8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGFA regulation revealed that XBP-1(S), a UPR-inducible transcription factor, bound to two regions on the VEGFA promoter, and analysis of XBP-1 null mouse embryonic fibroblasts revealed that it contributes to VEGFA expression in response to ER stress. ATF4, another UPR-inducible transcription factor, also binds to the VEGFA gene, although its contribution to VEGFA transcription appeared to be fairly modest. We also found that VEGFA mRNA stability is increased in response to UPR activation, via activation of AMP kinase, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the mRNA levels, we found that VEGFA protein is secreted at levels as high as or higher than that achieved in response to hypoxia. Conclusions and Significance Our results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. It also regulates VEGFA expression at transcriptional, post-transcriptional and post-translational levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer.

Endoplasmic Reticulum (ER) Stress and Hypoxia Response Pathways Interact to Potentiate Hypoxia-inducible Factor 1 (HIF-1) Transcriptional Activity on Targets Like Vascular Endothelial Growth Factor (VEGF)

Background: The UPR and HIF signaling pathways are cytoprotective responses activated by inadequate cellular environments. Results: These pathways can cooperate to increase the expression of shared targets like VEGF. Conclusion: The UPR enhances HIF-1 transcriptional activity. Significance: The simultaneous activation of both pathways in tumors may result in greater vascularization. Cells respond to suboptimal microenvironments by activating stress signaling pathways, like the unfolded protein response and hypoxia-induced transcription factors HIF-1/2, to restore homeostasis. Both cytoprotective pathways have been well studied in isolation at the biochemical and molecular levels. Mounting evidence reveals that they can be activated simultaneously in tumor cells and, likely, in other tissues experiencing inadequate microenvironments and that they share some transcriptional targets, like the proangiogenic factor VEGFA. However, the potential interaction between these pathways is poorly understood. Cell culture experiments revealed that as a consequence of unfolded protein response activation, ATF4 bound to the human VEGFA promoter and activated its transcription, whereas HIF-1 did so in response to hypoxia. When both pathways were activated together, VEGFA transcripts were induced to a higher level than when either stress was applied alone. Surprisingly, this was not due to the combined actions of the stress pathway-specific transcription factors. Instead, we found that endoplasmic reticulum stress potentiated HIF-1 activity to transactivate VEGF expression as well as another well characterized target, BNIP3. These data reveal an unexpected interaction between two important cytoprotective responses that are likely to have significant consequences in environmentally compromised tissues and tumor cells.

The lymph node microenvironment and its role in the progression of metastatic cancer

Lymph nodes are initial sites for cancer metastasis in many solid tumors. However, their role in cancer progression is still not completely understood. Emerging evidence suggests that the lymph node microenvironment provides hospitable soil for the seeding and proliferation of cancer cells. Resident immune and stromal cells in the lymph node express and secrete molecules that may facilitate the survival of cancer cells in this organ. More comprehensive studies are warranted to fully understand the importance of the lymph node in tumor progression. Here, we will review the current knowledge of the role of the lymph node microenvironment in metastatic progression.

Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice

An alternate route for metastatic cells Metastatic tumor cells are thought to reach distant organs by traveling through the blood circulation or the lymphatic system. Two studies of mouse models now suggest a hybrid route for tumor cell dissemination. Pereira et al. and Brown et al. used distinct methodologies to monitor the fate of tumor cells in lymph nodes. They found that tumor cells could invade local blood vessels within a node, exit the node by entering the blood circulation, then go on to colonize the lung. Whether this dissemination route occurs in cancer patients is unknown; the answer could potentially change the way that affected lymph nodes are treated in cancer. Science, this issue p. 1403, p. 1408 In mice, tumor cells can metastasize to distant organs by entering blood vessels within the local lymph node. Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognoses, and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma, or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in cancer patients remains to be determined.

Angiopoietin-4 increases permeability of blood vessels and promotes lymphatic dilation

The angiopoietin (Ang) ligands are potential therapeutic targets for lymphatic related diseases, which include lymphedema and cancer. Ang‐1 and Ang‐2 functions are established, but those of Ang‐4 are poorly understood. We used intravital fluorescence microscopy to characterize Ang‐4 actions on T241 murine fibrosarcomaassociated vessels in mice. The diameters of lymphatic vessels draining Ang‐4‐ or VEGF‐C (positive control)‐expressing tumors increased to 123 and 135 μm, respectively, and parental, mock‐transduced (negative controls) and tumors expressing Ang‐1 or Ang‐2 remained at baseline (~60 μm). Ang‐4 decreased human dermal lymphatic endothelial cell (LEC) monolayer permeability by 27% while increasing human dermal blood endothelial cell (BEC) monolayer permeability by 200%. In vivo, Ang‐4 stimulated a 4.5‐fold increase in tumor‐associated blood vessel permeability compared with control when measured using intravital quantitative multiphoton microscopy. Ang‐4 activated receptor signaling in both LECs and BECs, evidenced by tyrosine kinase with Ig and endothelial growth factor homology domains‐2 (TIE2) receptor, protein kinase B, and Erk1,2 phosphorylation detectable by immunoblotting. These data suggest that Ang‐4 actions are mediated through cell‐type‐specific networks and that lymphatic vessel dilation occurs secondarily to increased vascular leakage. Ang‐4 also promoted survival of LECs. Thus, blocking Ang‐4 may prune the draining lymphatic vasculature and decrease interstitial fluid pressure (IFP) by reducing vascular permeability.—Kesler, C. T., Pereira, E. R., Cui, C. H., Nelson, G. M., Masuck, D. J., Baish, J. W., Padera, T. P. Angiopoietin‐4 increases permeability of blood vessels and promotes lymphatic dilation. FASEB J. 29, 3668‐3677 (2015). www.fasebj.org

Murine chronic lymph node window for longitudinal intravital lymph node imaging

Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice. The CLNW is custom-made from titanium and incorporates a standard coverslip. It allows stable longitudinal imaging without the need for serial surgeries while preserving lymph node blood and lymph flow. We also describe how to build and use an imaging stage specifically designed for the CLNW to prevent (large) rotational changes as well as respiratory movement during imaging. The entire procedure takes approximately half an hour per mouse, and subsequently allows for longitudinal intravital imaging of the murine lymph node and surrounding structures for up to 14 d. Small-animal surgery experience is required to successfully carry out the protocol.

Methicillin-resistant Staphylococcus aureus causes sustained collecting lymphatic vessel dysfunction

Impairment in lymph flow caused by methicillin-resistant Staphylococcus aureus toxins persists after skin and soft tissue infections clear. Lymphatics limp along after MRSA Lymphedema is associated with skin and soft tissue infections, and both can be recurring, causing continual suffering in affected patients. To better understand the relationship between bacterial infections and lymphedema, Jones et al. used intravital imaging to examine the lymphatics of mice infected with MRSA. They observed lymphatic muscle cell death, which led to prolonged dysfunction months after the bacteria had been cleared. In vitro experiments with human cells indicated that bacterial toxins were responsible for damaging the lymphatic muscle cells, suggesting that the toxins could be targeted in patients to interrupt this brutal cycle. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality worldwide and is a frequent cause of skin and soft tissue infections (SSTIs). Lymphedema—fluid accumulation in tissue caused by impaired lymphatic vessel function—is a strong risk factor for SSTIs. SSTIs also frequently recur in patients and sometimes lead to acquired lymphedema. However, the mechanism of how SSTIs can be both the consequence and the cause of lymphatic vessel dysfunction is not known. Intravital imaging in mice revealed an acute reduction in both lymphatic vessel contractility and lymph flow after localized MRSA infection. Moreover, chronic lymphatic impairment is observed long after MRSA is cleared and inflammation is resolved. Associated with decreased collecting lymphatic vessel function was the loss and disorganization of lymphatic muscle cells (LMCs), which are critical for lymphatic contraction. In vitro, incubation with MRSA-conditioned supernatant led to LMC death. Proteomic analysis identified several accessory gene regulator (agr)–controlled MRSA exotoxins that contribute to LMC death. Infection with agr mutant MRSA resulted in sustained lymphatic function compared to animals infected with wild-type MRSA. Our findings suggest that agr is a promising target to preserve lymphatic vessel function and promote immunity during SSTIs.

Lymph node effective vascular permeability and chemotherapy uptake

Lymph node metastases are a poor prognostic factor. Additionally, responses of lymph node metastasis to therapy can be different from the primary tumor. Investigating the physiologic lymph node blood vasculature might give insight into the ability of systemic drugs to penetrate the lymph node, and thus into the differential effect of therapy between lymph node metastasis and primary tumors. Here, we measured effective vascular permeability of lymph node blood vessels and attempted to increase chemotherapy penetration by increasing effective vascular permeability.

Growth and Immune Evasion of Lymph Node Metastasis

Cancer patients with lymph node (LN) metastases have a worse prognosis than those without nodal disease. However, why LN metastases correlate with reduced patient survival is poorly understood. Recent findings provide insight into mechanisms underlying tumor growth in LNs. Tumor cells and their secreted molecules engage stromal, myeloid, and lymphoid cells within primary tumors and in the lymphatic system, decreasing antitumor immunity and promoting tumor growth. Understanding the mechanisms of cancer survival and growth in LNs is key to designing effective therapy for the eradication of LN metastases. In addition, uncovering the implications of LN metastasis for systemic tumor burden will inform treatment decisions. In this review, we discuss the current knowledge of the seeding, growth, and further dissemination of LN metastases.

Breast cancer metastasis through the lympho-vascular system

Breast cancer metastasizes through the lymphovascular system to the regional lymph nodes in the axilla and to both visceral and non-visceral sites. Renewed interest in the route by which tumor cells gain access to blood and lymphatic capillaries are the subject of research at mechanical, anatomic, pathologic, genetic, epidemiologic and molecular levels. Two papers presented at the 7th International Symposium on Cancer Metastasis in San Francisco showed tumor cells entering the systemic circulation through the sentinel lymph node. This information challenges the current paradigm where clinicians believe that access is gained through intra- and peri-tumoral blood vessels and that metastasis to axillary lymph nodes is an interesting epi-phenomenon. The sentinel lymph node era has changed the modern surgical approach to the axilla and the basis of this change is summarized in this paper. A new approach to the management of axillary metastases after systemic therapy relies on determining whether there is a complete pathologic response; if no tumor is found in the previously biopsied node, a complete axillary lymph node dissection may be avoided. African American women seem to inherit a trait from West African ancestors and tend to develop more lethal types of breast cancer. These tumors may have a molecular machinery that enhances their ability to metastasize to visceral sites and future research may unearth the mechanisms for this phenomenon.