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Eukaryon

Inhibition of Brain Metastases Through the STING Pathway

Marisol Carreon, Rachel Domijancic, Schuyler Kogan, Rebecca Lynch
Departments of Biology and Neuroscience
Lake Forest College
Lake Forest, Illinois 60045

Brain metastasis, the invasion of the central nervous system by cancer cells from other tissue, is common in many forms of cancer despite the brain’s powerful defenses. This development can drastically decrease the patient’s prognosis, as the cancer cells can easily disturb the fragile environment of the brain. Overactivity of the stimulator of interferon genes (STING) pathway is involved in brain metastasis of breast cancer. This pathway is stimulated by DNA released by unstable chromosomes and by Cx43 gap junction interactions between cancer cells and astrocytes, leading to the release of interferon (IFN) compounds that can promote cancer success (3,5). STING may be a powerful target for preventative therapies, but the findings relating to breast cancer are not generalizable to other cancer types without further research. We intend to investigate this by first measuring STING expression in brain metastatic cancer cells of multiple types. Then, to form a causal link, we will experimentally inhibit or enhance the STING pathway in cancer models and measure the resulting metastasis. Finally, to determine aspects of the pathway shared between cancer types, we will specifically manipulate DNA detection, Cx43 gap junctions and IFN release. Our findings will help illustrate shared or varying roles of the STING pathway in brain metastasis, and potentially help in the development of general therapies to prevent brain metastasis.

 

 

Background

 

                Cancer, the unregulated proliferation of abnormal cells, comes in many forms which can pose a serious threat to human health.  Tumors can form spontaneously, or from inheritable gene mutations or environmental factors (1). There are five different types of cancers: carcinoma (starts in skin or organ lining tissues), sarcoma (starts in connective or supportive tissues), leukaemia (blood tissues), lymphoma (immune system), and brain and spinal cord (40). Cancer has varying negative effects on the body, depending on where it is located. For example, it can increase or decrease the number of red blood cells circulating the body, create blockages, weaken our immune system, create hormonal imbalance, or cause damage to surrounding tissue (41). There are some treatments available, such as chemotherapy, to try and eliminate cancers; however, cancer can come back and spread againtime if there are traces of cancer cells left behind, or if the cancer becomes resistant to treatment (39).

However, humans are not completely defenseless. The CNS has several powerful, unique defenses against invading cells. Even if cancer can manage to pass the blood-brain barrier (BBB), the cancer cells must still survive against reactive astrocytes and a different extracellular environment to the external tissue (3, 4). Despite these defense mechanisms, up to 40% of advanced cancers can spread to the brain (1). There are multiple different molecular mechanisms that can possibly let cancer cells metastasize. One possible mechanism for the metastasis of cancer cells is through chromosomal instability (5). Chromosomal instability leads to an increase in cytosolic DNA that increases the activation of the STING pathway (5). Research has indicated that breast cancer cells overexpress COX2. This promotes the production of MMP1, an enzyme that breaks down extracellular matrices, weakening the junctions of the BBB and allowing passage, as well as compounds that enhance tumor cell survival (4). In order for cancer cells to survive in different microenvironments, different cancer cells, like breast and melanoma, have been shown to both co-oxidize acetate and glucose  to make acetyl-CoA from the citric acid cycle for energy (14). In a study done by Tominaga et al., they found that breast cancer cells used extracellular vesicles to send in miR-181c to promote the breakdown of the blood brain barrier through the PDPK1 gene in order to metastasize to the brain (15). This shows that there are many mechanisms that cancer cells are able to use in order to spread into the brain and cause more harm; however, there is a commonality in the pathway that cancer cells from different origins use in order to metastasize.

One pathway involved in successful brain metastasis that sparked our interest was the stimulator of interferon genes (STING) pathway (3, 5). The primary function of the STING pathway is to detect and respond to cytosolic DNA, often to generate an immune defense against viral interference. However, STING also often triggers a response to the activity of cancer cells which can have significant effects on the success of metastasis. Some evidence suggests that STING’s activity can result in inflammation that promotes cancer growth or invasion (6, 7). Upon activation, STING phosphorylates NF-κB, MAPK and IRF3, leading to the regulation of pro-inflammatory genes such as IL-6 (8). Evidence that supports STING promotes cancer growth through an inflammatory response is also the case in brain metastasis, where the inhibition of STING has significantly reduced invasion (3, 5). However, other research has demonstrated that STING has antitumor properties, and it appears that the pathway’s relationship to cancer growth and metastasis depends on many factors, including the invaded tissue and the form of tumor (9-11). Not only that, but the reduction of STING activity, either experimentally or because of cellular dysfunction, can potentially allow for both viral infection and tumorigenesis (12, 13).

One possible reason for STING promoting metastasis into the CNS specifically is that it is involved in an interaction between cancer cells and reactive astrocytes. By forming gap junctions with the astrocytes, the cancer cells can secrete the secondary messenger Cyclic guanosine monophosphate -adenosine monophosphate (cGAMP) into the astrocytes, activating STING and triggering cytokine production to promote tumor survival and growth (3). This indicates a potential therapy that could block STING’s effect on invasion of the CNS without the risk of serious side effects. Based on STING’s role in cancer and metastasis, we will focus on trying to figure out the mechanism of how it successfully promotes metastasis, and on what therapeutic advances can be made when inhibited. Our gap in knowledge, based on previous research, is whether or not we can generalize STING’s relationship to brain metastasis to multiple types of cancer. Our hypothesis is that STING contributes to brain metastasis in varying ways in different forms of cancer.

               

Relevance

 

                Broader Relevance: There is an estimated 200,000 to 300,000 people per year that suffer from brain metastatic cancers and 700,000 Americans with brain tumors. On average, 10-20% of metastatic brain tumors arise as a single tumor. People ages 45-64 have an increased risk for developing brain metastasis, and those that are older than 65 have the highest risk. Lung, breast, and skin cancer are the most common types of cancer that can develop into brain metastasis. The average survival rate for all malignant tumor patients is only 34.7%, specifically, 33.8% being for males and 36.4% for females (38).

By researching the STING pathway in cancer cells, especially those with the ability to metastasize into the brain, we may identify new target sites in order to develop new therapeutic approaches for cancer patients. If STING inhibition can reduce brain metastasis, cancer could be contained at one site and be easier to target with chemotherapy. Our research may also help to develop potential preventative measures and screening tests in order to detect cancer at earlier stages before it has a chance to pass through the blood brain barrier. With the use of assays and immunofluorescence tests, medical professionals can also measure STING within cancer patients to get a visual of where the cancer is and if it has started to spread.

                Intellectual Merit: This series of experiments can help us further our knowledge of cancer and the ways it is able to metastasize to other parts of the body, specifically to the brain. The STING pathway is a relatively new key component of cancer research, and previous studies have shown that when it is present cancer cells have a higher tendency to metastasize past the blood brain barrier. Researching what tests and genes are able to measure and manipulate STING activation can help us understand the pathway at a deeper level. It can also help  determine the role of STING in cancer cells such as in lung, breast, and human melanoma, which are the most common types of cancer. In the future, findings from this research could help scientists to better understand mechanisms of brain metastasis and may lead to new targeted therapeutic approaches for cancer patients.

 

Specific Aims

 

                Our hypothesis is that STING contributes to brain metastasis in varying ways in different forms of cancer. In order to support our hypothesis, we will try to generalize the findings about STING’s role in brain metastasis to different common cancer cell types. STING has been implicated to facilitate brain metastasis of breast cancer, but its relationship in other forms of cancer varies, so more research is necessary before connecting STING to brain metastasis in general (3,5,8).

 

To test this, we will:

  1. Induce brain metastasis from different forms of cancer (breast, lung, and skin) and measure the expression of STING: To determine if STING is expressed not only in cancer cells in general, but also in metastatic cancer sourced from different origins of cancer like breast, lung, and skin, we will develop models that will help isolate non-metastatic and metastatic cells both in vivo and in vitro, and determine which techniques we will use that effectively measure STING.
  2. Inhibit STING with shRNA and assess changes in brain metastasis: To inhibit STING, we will use shRNA, an antagonist, and a genetic knockout mouse. We will then observe the expression of TBK1, IRF3, and STAT1 to establish expression of STING. We will also observe levels of brain metastasis.
  3. Enhance STING with an agonist and assess changes in brain metastasis: To enhance STING expression, we will use cyclic dinucleotides and an agonist. We will then observe the expression of TBK1, IRF3, and STAT1 to establish expression of STING. We will also observe levels of brain metastasis.
  4. Selectively alter different elements of the STING pathway and assess changes in brain metastasis: To identify which aspects of the STING pathway are significant to brain metastasis and to highlight potential cell-type-specific features, we will individually inhibit and enhance cGAMP, Cx43 and IFN and measure the resulting changes in metastasis.

 

Research Methods and Design

 

  1. Induce brain metastasis from different forms of cancer (breast, lung, and skin) and measure the expression of STING

 

Rationale: The  STING pathway is active in cancer cells that are able to pass through the blood brain barrier and metastasize into the brain (8). In the past, it has been shown the STING plays an important role in breast cancer; however, in this experiment, we will also use lung cancer and human melanoma. In order to determine which cells have higher metastatic activity, we will use both in vivo and in vitro models to measure STING in both the positive and negative controls for comparison. By making sure that STING is present in the cancer cells, we can proceed to inhibit and enhance STING in the next series of experiments.

 

1A. Model brain metastasis in vitro and observe STING expression in metastatic cells:

We will use three types of cell lines to model brain metastasis: human non-small cell lung cancer (NSCLC) cell lines, human breast cancer cell lines, and human melanoma cell lines (4, 16, 37). We can model brain metastasis in vitro by replicating the BBB using primary cultures of monkey brain capillary endothelial cells, brain pericytes, and astrocytes (15). We will know that the model works based on previous research done (15). Each cell line will begin on the upper chamber of the in vitro BBB model and will be left to infiltrate for 2 days. We will count how many cells pass through to the other side as a measure of metastasis (15). We will consider cancer cells passing through this artificial barrier as metastatic, while cells remaining in the upper chamber will be non-metastatic.

When treated with dsDNA, IRF3 translocation/phosphorylation into the nucleus will demonstrate normal activation of the STING pathway in all cell lines (13). Co-incubation of cancer cells from metastatic and non-metastatic groups will demonstrate any Cx43-dependent phosphorylation of TBK1 and IRF3. This will show STING activation, since cGAMP binding to the pathway triggers phosphorylation and activation of TBK1 and IRF3 (3). Immunofluorescent staining and western immunoblotting will visualize STING activation by testing for TBK1, IRF3, and STAT1 (5). Flow cytometric assays of STAT1 phosphorylation and qPCR array analysis of cGAMP expression will also measure STING pathway activation (8,9). Within-subject t-test will be used to analyze the upper and lower cancer cells and ANOVA will be used to compare the three different cancer cell lines.

Predictions: The metastatic cancer cell lines that pass through the artificial barrier will have normal Cx43-dependent phosphorylation of IRF3 and TBK1 when treated with the dsDNA, which will confirm that STING is activated. To visualize this activation, immunoblotting will show that STING is present within the cancer cells. cGAMP, will also be prevalent in the metastatic cancer cells, which will further confirm that STING is present in them, as compared to the non-metastatic groups of cancer cells which will be negative. Immunofluorescent staining of the metastatic cancer cells will be brighter than the non-metastatic cancer cells because of the larger presence of STING.

 

1B. Model brain metastasis in vivo and observe STING expression in metastatic cells:

We will create in vivo metastatic models by transplanting these NSCLC cell lines that have passed into the lower chamber of the BBB in vitro model into athymic mice (laboratory mice lacking thymus glands and without T cells) through a tail vein and through intra-left ventricular inoculation (16). For a control group, we will use mice injected through the tail vein and through the intra-left ventricular inoculation with the non-metastatic cells that were left behind in the upper chamber. We can monitor brain metastasis while the model rodents are alive by observing behavioral impairment, such as physiological and metabolic changes and loss of coordination, by the formation of tumors of over .5cm in diameter, or by using intraperitoneal luciferin injections (13,15,17). After the development of brain tumors, or after 3 months, we will sacrifice the mice, and perform bioluminescence techniques that will allow for measurement of the levels of metastasis. The bioluminescence marker, luciferase, will be used (24, 25). The brains will then be extracted and minced from both the positive and negative controls. We will then culture the brains with 18 ml of culture medium in flasks and collect the cells attached to the bottom of the flasks (16). Afterwards, we will stain the cells with human specific monoclonal antibody (anti-integrin beta1 subunit) and measure by flow cytometry (16). We will also measure STING presence through immunofluorescence staining and western immunoblotting for TBK1, IRF3, and STAT1 (5). Within-subject t-test will be used to analyze the upper and lower cancer cells and ANOVA will be used to compare the three different cancer cell lines.

 

 Predictions: The mice with the metastatic cancer cells will develop large tumor masses in their brains within the 3-month period and when stained with human specific monoclonal antibody in the flow cytometry, the STING expression will be present more than the non-metastatic mice. Mice injected with the negative control will take longer to develop visible tumors. Immunofluorescence staining and western immunoblotting results will be similar to that of aim 1A.

 

  1. Inhibit STING with shRNA and assess changes in brain metastasis

 

Rationale: STING has been shown to play different roles in different types of cancer. shRNA has been shown to successfully inhibit certain pathways (18). CCCP is an effective antagonist of the STING pathway as it  suppresses the phosphorylation of TBK1 and IRF3 (19, 20, 21). By inhibiting STING with the previously mentioned methods, the effects on three specific cell types will be visible. Knockdown mice will also be used to observe the effects of STING inhibition. Depending on the effects that these methods have on brain metastasis in the different cell lines, inhibition of STING could be a potential therapeutic target.

 

2A. Genetic inhibition of the STING pathway:

MDA-MB-231, 4T1, and H2030 with shRNA knockdown of STING serve as the in vitro model of genetic inhibition (5). Cell lines will be injected with shRNA in order to down-regulate the expression of STING. The control groups will be injected with a control vector, TCR1. Knockout mice will be used as an in vivo model for STING inhibition. There will be two conditions, WT and STING-/-. Wild type littermates will be used as the control condition. Sixteen mice will be used in total. STING inhibition will be determined by measuring levels of TBK1, IRF3, and STAT1 using western blotting and immunohistochemistry techniques (18). Bioluminescence techniques will allow for measurement of the levels of metastasis. The bioluminescence marker, luciferase, will be used (24, 25). Statistical analysis will be done using independent samples t-test and an ANOVA test.

Predictions: Based on previous research, we expect that the expression of TBK1, IRF3, and STAT1 will be much lower in the shRNA and knockout conditions. The levels of bioluminescence will also be much lower in STING inhibited conditions. The level of immunofluorescence for TBK1, IRF3, and STAT1 will also be much lower for the STING knockout and shRNA condition. Statistical analysis will be done using independent samples t-test and an ANOVA test.

 

2B. Chemical inhibition of the STING pathway:

 in vitro models will be the same as in the genetic inhibition. The STING antagonist carbonyl cyanide 3-chlorophenylhydrazone (CCCP) will be used to inhibit the STING pathway (19, 20, 21). CCCP inhibits the STING pathway by suppressing the phosphorylation of TBK1 and IRF3 (19, 20, 21). The control group will be tested using PBS. STING inhibition will be determined by measuring levels of TBK1, IRF3, and STAT1 using western blotting and immunohistochemistry techniques (18). Bioluminescence techniques will allow for measurement of the levels of metastasis. The bioluminescence marker, luciferase, will be used (24, 25). Statistical analysis will be done using independent samples t-test and an ANOVA test.

Predictions: Based on previous research, we expect that the expression of TBK1, IRF3, and STAT1 will be much lower in the CCCP condition. The levels of bioluminescence will also be much lower in the STING inhibited condition. The level of immunofluorescence for TBK1, IRF3, and STAT1 will also be much lower for the CCCP condition.


  1. Enhance STING with an agonist and assess changes brain metastasis

 

Rationale: The STING signaling pathway is activated by cyclic dinucleotides (CDNs), and we plan on altering the microenvironment and enhancing the number of CDNs by 3 different agonists, which previous literature has shown to enhance STING. (26, 27). The treatment of more CDNs via has shown to decrease tumor metastasis and size. The more CDNs that STING senses, the greater the downstream responses STING will elicit. These responses include an increase in type 1 interferon (IFN), programmed death ligand 1 (PD-1) and proinflammatory cytokine production (28). Another way in which we plan on increasing tumor activity is by using a STING agonist, MK-1454. MK-1454 stimulates STING to increase immune responses (29). These tumor suppression factors will increase macrophage activity and tumor cell death, thus decreasing tumor metastasis. For both subaims, a transgenic mouse model will be used and will be similar to the previous aim, but instead there will be a WT and knockin mouse to enhance STINGs activity. By utilizing different CDNs and agonists for the STING pathway and for multiple cancer cell lines, we could observe a relationship between these two factors and observe which downstream effects resulted in a more pronounced decrease in metastatic rate. This could lead to the discovery of more effective therapeutic methods. We intend to analyze the efficacy of using CDNs to increase STING activity among the chosen cell lines to suppress tumor growth of the cancers most likely to metastasize to the brain.

 

3A. Manipulate the tumor microenvironment by an agonist of the STING signaling pathway via cytosolic cyclic dinucleotides (CDNs) in vivo and in vitro:

in vitro and in vivo models will be the same as those used in the previous aims. We will use three conditions with four mouse models in each condition: a control group, 2’3’ c-di-AM(PS)2(Rp,Rp) and 2’3’-cGAMP CDN treatments. The control groups will be treated with PBS instead of drugs. The mice will all have received transplantable metastatic tumor models of human breast cancer from cell line MDA-MB-23. The conditions will receive either 20 μg of the drug or of PBS.  To measure macrophage activity, we will run a Macrophage Inflammatory Assay to assess activation of macrophage subpopulations (M1 and M2) (27, 28, 30). The assay will be quantified and compared to the control using by an independent-samples t-test, and the different conditions will be compared using an ANOVA test.

Predictions: The newest STING agonist, 2’3’ c-di-AM(PS)2(Rp,Rp), will be most effective at limiting metastasis in cell lines MDA-MB-231 and H2030 (27). This treatment could possibly lead to an increase in metastasis with the breast cancer cell line 4T1 due to its dual role in breast cancer, as previously mentioned in Aim 2.

 

3B. Evaluate an optimal dose for STING agonist MK-1454 using in vitro and in vivo models:

Cell lines MDA-MB-231, 4T1 and H2030 will be used as an in vitro model to assess what the optimal dosage of another STING agonist, MK-1454, would best suit each cancer cell line. As informed by previous literature, there will be three different concentrations of MK-1454: low, moderate, and high (29). The conditions will then be transferred into athymic mice, as used before. The mice will then be given the vaccine at different intervals of time: day 1, day 10 and day 20. We will examine the efficacy of the different concentrations of MK-1454 and at what part of the treatment the drug was most effective. Programmed cell death and metastasis of the tumor will be assessed and quantified as before.

Predictions: MK-1454 will be most effective in the Day 1 and low-moderate concentration group. This treatment could be effective in helping current phase 1 clinical trials so that the concentration and the stage of the cancer could be properly paired.

 

  1. Selectively alter different elements of the STING pathway and assess changes in brain metastasis

 

Rationale: The interaction between reactive astrocytes and cancer cells through the Cx43 gap junction is one of the stronger links between brain-specific effects on metastasis and the STING pathway. However, this has primarily been tested with breast cancer cell lines (3). By manipulating the Cx43 gap junction in multiple different types of cancer, we intend to investigate the degree that this intercellular interaction can be generalized. We will also be manipulating two other elements of the STING pathway considered to be essential: cGAMP signalling and interferon production (4,6). Our focus in this aim may be determined by our findings in aims 1 through 3, as unique reactions to STING in some cell lines could be explained by directly manipulating these specific elements.

4A. Selectively inhibit cGAMP, Cx43 and IFN individually:

in vitro and rodent models will be created using the same methods as in the previous three aims. We will use four groups of five subjects from each model type: a control group, and groups with inhibited cGAMP, Cx43 and IFN function. The control groups will be treated with PBS rather than  drugs. The cGAMP- groups will be treated with the cGAS inhibitor PF-06928215, which prevents the release of cGAMP (31). The Cx43- groups will be treated with the channel blocking peptide Gap19 (32). The IFN- groups will be treated with interferon inhibitor (IFI), blocking the inflammatory effects of IFN (33). As an alternate manipulation, we will also produce knockout mice targeting the cGAS, GJA1 and IFNA1 genes. All four groups will be be tested with a western blot to assess the level of STING expression after each manipulation (18). The same procedure as aims 2 and 3 will be used to measure brain metastasis, each of the three experimental groups will be compared to the control group using an independent-samples t-test, and the three cell lines will be compared to each other using an ANOVA test.

Predictions: Based on previous research in breast cancer cell lines, we expect that the inhibition of Cx43, cGAMP or IFN should each independently reduce brain metastasis. If metastasis is conserved in any condition for any cell line, it could indicate that metastasis is mediated by an alternate mechanism that is either cell type-specific (such as an alternate gap junction or signalling molecule) or nonspecific (such as increased chromosomal instability). Increased metastasis in the inhibited groups for any cell line could indicate a potential cell type-specific therapeutic possibility for STING, which could be more easily distinguishable in subaim B.

 

4B. Selectively enhance cGAMP, Cx43 and IFN individually:

The same set of models will again be used in four groups, with the experimental groups now manipulated to have enhanced cGAMP, Cx43 or IFN activity. The cGAMP+ groups will be enhanced using the cGAS agonist DSDP, which will increase cGAMP release (34). The Cx43+ groups will be treated with the peptide AAP10, which increases expression of Cx43 (35). The IFN+ group will be treated with IFN inducer RNase L, which enhances production of IFN (36). As an alternate manipulation, we will also produce mice with genetic overexpression of the cGAS, GJA1 and IFNA1 genes. The same measurements of STING expression and metastasis will be used as in the previous subaim.

Predictions: We expect that enhancement of of Cx43, cGAMP or IFN should each independently increase brain metastasis relative to the control. If metastasis is unaffected in some cell types, it could indicate that the manipulated element does not play the same role, or that there is some sort of limiting factor or saturation point. Decreased metastasis in the enhanced groups for any cell line could indicate a potential cell type-specific therapeutic possibility for STING.

 

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