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Improvements in Small Animal Dosimetry: CIX3 Irradiator Characterization, Novel Phantom Investigation, and Shepherd Cs-137 Irradiator Dose Uniformity Analysis
No full textProject 1 (Chapter 2): X-Strahl CIX3 CharacterizationPurpose: In Fall 2021 Duke University purchased an Xstrahl CIX3 Cabinet Xray
Irradiator. A characterization of this machine was performed to determine its
dosimetry characteristics and best practices for radiobiological studies at Duke.
Basic irradiator acceptance tests were expanded on to fully characterize this new
machine. Two unique aspects of this machine were of particular interest. First a
unique (non-uniform) filter design was investigated to determine if it has unintended
side effects on field conformity. Second the lack of accessible cable ports
made thermoluminescent dosimeters (TLDs) the primary dosimeters for experiments.
For this reason additional investigations were conducted characterizing
TLDs. Materials and Methods: To assess the dosimetric properties of this new machine
the following parameters were investigated: output consistency, beam quality,
field uniformity, and exposure rates. Output consistency was measured by
comparing expected and observed max energy in kVp using a Piranha X-ray multimeter.
Beam quality was measured as half value layer of aluminum and compared
to expected results from Spekcalc. Spekcalc was used to determine energy
fluence and mean energy of the spectrum. Field uniformity was assessed using
Gafchromic ™ EBT3 film on an Epson Expression 10000 XL scanner with lateral
response artifact correction factors and film calibrated using a NIST traceable 0.18
cc ion chamber. Exposure rates were characterized using a NIST traceable 0.18cc
ion chamber and varying filtration, tube energy (kVp), and tray position for all
available configurations. TLD energy response, positional response, and batch correction factor techniques
were characterized on this machine. Energy response was determined by
irradiating TLD’s to a range of energies (70-300 kVp) and the charge response reliv
ative to the exposure (50 R) received was determined. Positional response in the
field was investigated using the Duke Radiation Dosimetry Laboratory (DRDL)
TLD holder and Gafchromic ™ EBT3 film. The relative exposure each TLD received
was determined and compared to the ion chamber exposure. Results: The CIX3 had consistent energy output as measured by max energy conformance. The average difference from input voltage to output voltage was
0.84 % with the worst being 1.6 % (150 kVp, 1 mm copper Filtration). Theoretical
estimations (Spekcalc) of the beam quality had good agreement with measured
half value layers (using Piranha) with an average difference of 1.36 % and worst
error of 2.99 % across the energy ranges sampled (50-150 kVp). Field uniformity
results indicated general conformance to machine data (90 % within 25.9 cm diameter
field) but some non-uniformities were identified. Areas of higher dose
(105-110%) relative to the center were observed in the upper right quadrant of the
field (from beam eye perspective). TLD energy response followed expected over-response in lower energy ranges,
reducing to a normal response as energy increases. The highest over response was
at 70 kVp, a 15 % over response compared to 300 kVp (the max energy of the
CIX3). The film study to determine TLD positional response determined there
were unequal exposures to the 50 TLDs. The trend observed an increase in exposure
consistent with the field uniformity results in which the dose relative to the
center of the field increased by 5-10 % towards the upper right quadrant. This
effect was more pronounced at the lower energy level sampled (90 kVp).
Conclusions: The full characterization of the CIX3 was very important to understand
the nuances of the new machine. Conformance in beam quality results
gave good indications that the machine is operating as designed and radiobiological
studies expect to have consistent results between experiments under like
conditions. The unique field uniformity results observed could help inform future
experimental planning. More importantly it is an extremely important finding for
TLD dose calibration. Since TLDs experience at most 6-7 % over exposure compared
to the ion chamber it is important to use these findings in future calibrations.
Project 2 (Chapter 3): Dose Depth of Small AnimalWater Phantom
Purpose: Much is understood about the midpoint dose estimation of small animal
phantoms and it has been the focus of DRDL to conduct dosimetry using this
value. However, little data was available on the dose depth profile of small animal
phantoms. This investigation sought to fill in that dose depth data, compare doses
under varying experimental conditions in order to fully understand how the dose
is distributed along the beam axis for small animal phantoms.
Materials and Methods: A small animal water phantom (50 cc water vial) was
characterized on the CIX3 using Gafchromic ™ EBT3 film. Film was calibrated
using a NIST traceable 0.18 cc ion chamber. The phantom was irradiated with
strips of film (15 total) varying filtration and whether a backscatter plate (uniform
piece of acrylic) was included. The film was scanned and analyzed using Film QA
software and aggregated dose depth profiles were determined using R Studio and
Excel.
Results: The dose depth of the phantom was characterized with a coefficient
of variation of about 2-3 % across all depths and configurations. The inclusion of
the backscatter plate improved the dose uniformity by an average of 2.14 % with
most improvements coming from the bottom half of the phantom (closest to the
backscatter plate). Average dose rates under each configuration were determined.
The midpoint dose rate was found to have good conformance (within 0.5%) to the
averaged dose rate across the depth. The dose rate increased by 33 % when using
the backscatter plate due to the increased backscatter spectrum and the inverse
square law effects.
Conclusions: This data gave increased certainty in using the midpoint dose
as a surrogate measure for whole body dose averages in small animal phantoms.
The improvement in dose uniformity when using the backscatter plate seems like a
promising addition to future experimental configurations. However in application
the size of the backscatter plate makes it unusable for large experiment samples
and it should be implemented only in specific studies as determined during the
dosimetry consult with DRDL.
Project 3 (Chapter 4): Novel Mouse Phantom Investigation
Purpose: In recent years DRDL developed a novel mouse phantom based on
feedback from researchers. This novel phantom flattened the mouse to mimic a
mouse laying on an irradiation platform, especially when sedated. This investigation
sought to determine if the novel ’flat’ phantom made of polymethyl methacrylate
(acrylic) (PMMA) had a significant difference in dosimetry when compared
with a standard cylindrical phantom made of soft tissue equivalent material.
Materials and Methods: To compare the dosimetric differences each phantom
a FLUKA Monte Carlo simulation was compared with experimental results from
TLDs on the CIX3. In the simulation the dose was compared using a midpoint
dose volume. In the experimental design TLDs were used to determine the dose
at the midpoint of each phantom. The dose rates were analyzed and compared to
determine if there was a significant difference.
Results: The Monte Carlo results indicated there were very slight differences
between phantom rates. The cylinder phantom had a dose rate of 1.83±0.038 Gy/min
while the flat phantom had a midpoint dose of 1.81 ± 0.045 Gy/min, 1.1 percent
lower than the cylindrical phantom. An unpaired t-test was performed to determine
if the samples were different and was found to give a p-value of 0.71
which gives a high probability that the sampled data are not significantly different.
The experimental results were found to be similar. The cylinder phantom had
a dose rate of 1.77 ± 0.05 Gy/min while the flat phantom had a midpoint dose of
1.81 ± 0.045 Gy/min, 2.25 percent higher than the cylindrical phantom. Again a
t-test was performed and these were determined to not be significantly different
(p = 0.31).
Conclusions: The flat phantom therefore is very similar to the dosimetry found
in the cylinder phantom. Small variations due to material properties, height of
phantom, scatter material, and attenuating material all balanced out to provide
dosimetry properties that are similar. This means that the cheaper to manufacture
flat phantom is just as good as the much more expensive cylinder phantom and
both can be used for small animal dosimetry.
Project 4 (Chapter 5): Shepherd Mk I - 68A Dose Uniformity
Purpose: In 2009 a study was conducted to determine the dose uniformity in
a Shepherd Mk I-68A Cs-137 irradiator. Since then vast improvements have been
made on film design and software to analyze scanned film with improved accuracy.
A follow up study was designed to revisit this previous characterization and
update the dose uniformity of the irradiator cavity using these new dosimeters
and techniques.
Materials and Methods: Gafchromic ™ EBT3 film was calibrated using a NIST
traceable 0.18 cc ion chamber on the Shepherd irradiator in each of the three positions
available. Large film sheets were then irradiated in a 2 mm acrylic holder
in all positions under rotating and non-rotating configurations. The scanned film
was analyzed using FilmQA software, R studio, and Excel to determine the dose
uniformity relative to the dose in the center.
Results: An ion chamber sample was compared to the film results and found
to be in good agreement (within 1 %) which indicated the film was appropriately
irradiated, scanned, and calibrated. Rotating dose distributions in positions 2 and
3 were nearly equivalent to manufacturer predicted isodose distributions with
noted discrepancies at the edges of the field. At a height of 15 cm to achieve dose
uniformity of 100 % ± 5 % the rotating tray has a usable radius of 7 cm from the
center in position 2 and 8 cm in position 3. Stationary dose distributions were compared
to previous uniformity and found to be in general agreement in the center.
However the isodose mapping previously characterized did not include a scanner
lateral response artifact correction factor which indicated a better uniformity than
what was found in this experiment. Position 1 results were similar to previous
dose distributions and most importantly confirmed the positioning of the source
in the chamber.
Conclusions: The updated dose uniformity data provides QA feedback as part
of a larger dosimetry program for Duke University. These results indicated that
this irradiator was and still is performing as expected and no mechanical failures
have caused a source to become misaligned or any major changes to the expected
dosimetry.</p
Assessing the Water Footprint of Electric Car Batteries – A Dive into the Water-Energy Nexus
No full textWater has been historically overlooked as a criterion when measuring the environmental impact of a project. This project aims to visualize the water impacts and risks associated with extracting three critical minerals commonly used in electric vehicle (EV) batteries (lithium, cobalt, and nickel) on behalf of Rivian – an EV manufacturing company. As EVs become increasingly popular, the demand for minerals and metals used in their production, such as lithium, cobalt, and nickel, has increased. The mining of these minerals often takes place in water-stressed areas, which can have negative environmental and social impacts. The goal of this project is to assess the water risks associated with mining these minerals and provide recommendations for a more sustainable supply chain.
The objectives of this project are to identify potential "hot spots" in the EV supply chain where water risks are most prevalent, evaluate the consumption of water from mining the three minerals, and provide recommendations to create a more sustainable supply chain.
We narrowed the supply chain to include an analysis of the mining of three critical EV battery raw materials – lithium, cobalt, and nickel. We researched the specific supply chains of these three minerals and found the geographic location of the top 10 mines by production, with some exceptions. These mines were then overlayed with water scarcity data from WRI’s Aqueduct tool. A dashboard was created to express these findings.
In the interest of transparency, we made sure to gather as much water consumption data as possible for the mining processes of lithium, cobalt, and nickel. Though we encountered some limitations during this process, such as differing functional units and definitions of water consumption/use, we did our best to create an informative table displaying our findings. We acknowledge that some of our sources lacked scientific confidence and our sample may not have been fully representative.
Potential supply chain hot spots - mines located in areas of high stress extremely high stress, or arid and low water use - were located for each mineral. For cobalt, the potential hot spots included the Murrin Murrin mine in Australia. For lithium, the potential hot spots include Sociedad Quimica y Minera de Chile and Albemarle’s Chile operations. It is also important to note the Greenbush Mine in Australia was located less than 10 miles from a location of high water scarcity and thus, was included in our potential hot spots. For nickel, the potential hot spots include Mount Keith Mine in Australia.
The broader ramifications of this work include the potential to promote more sustainable practices within the EV industry. By identifying potential "hot spots" in the supply chain where water stress risks are most present, this project provides a framework for developing a more sustainable supply chain. The recommendations provided in this project can help stakeholders in the EV industry to make more wholistic decisions about the environmental and social impacts of their production practices by including water consumption impacts.
This project highlights the need for greater attention to be paid to water scarcity within the EV supply chain. By analyzing the water risks associated with mining critical minerals for EVs and providing recommendations for a more sustainable supply chain, this project seeks to promote more responsible production practices within the EV industry. The findings of this project have the potential to inform future research and policy initiatives aimed at addressing the environmental and social impacts of EV production.
Moving forward, there is a need for more comprehensive data on water consumption and direct engagement with upstream suppliers to better understand the potential risks at these locations. Companies in the EV industry should also assess the sustainability of their supply chains on an individual level and explore alternative sources for critical minerals to reduce reliance on high-risk locations
The impact of lumbar alignment targets on mechanical complications after adult lumbar scoliosis surgery.
No full textPurposeThe purpose of this study was to determine the discriminatory ability of age-adjusted alignment offset and the global alignment and proportion (GAP) score parameters to predict postoperative mechanical complications.MethodsSurgical patients from the Adult Symptomatic Lumbar Scoliosis cohort were reviewed at 2 year follow up. Age-adjusted alignment offsets and GAP parameters were calculated for each patient. A series of nonlinear logistic regression models were fit, and the odds of mechanical complications were calculated. The discriminatory ability of the GAP score, GAP score parameters, and age-adjusted alignment offsets were determined plotting receiver operative characteristic (ROC) with the C statistic (AUC).ResultsA total of 165 patients were included. A total of 49 mechanical complications occurred in 41 patients (21 proximal junctional kyphosis and 28 pseudoarthrosis). The GAP score had no discriminatory ability in this cohort. Relative lumbar lordosis 15 degrees greater than ideal lumbar lordosis was associated with greater mechanical complications. A lumbar distribution index of 90% was associated with fewer mechanical complications compared to a lumbar distribution index of 65%. Age-adjusted offset alignment targets had no discriminatory ability to predict mechanical complications.ConclusionRadiographic alignment targets using either age-adjusted alignment target offset or GAP score parameters had minimal ability to predict mechanical complications in isolation. Mechanical complications following adult spinal deformity surgery are complex, and patient factors play a critical role. Clinical trial registeration This study was registered at ClinicalTrials.gov (number NCT00854828) in March 2009
Increasing Glaucoma Detection in Roatán, Honduras: The Value of Education and Intraocular Pressure Screenings
No full textGlaucoma is an ocular neuropathy that damages the optic nerve, leading to vision loss and blindness. Insidious in its presentation and difficult to diagnose, glaucoma is asymptomatic until irreversible harm is caused. Accounting for 12.6% of preventable blindness in Honduras, glaucoma inflicts considerable clinical, economic, and personal burdens on thousands of lives (Rosa et al., 2022). Early detection and treatment halves the risk of disease progression by lowering the intraocular pressure so that permanent harm to the optic nerve is avoided (Leskea et al., 2004). However, in Roatán, where access to ophthalmic care is lacking and awareness of the disease is scarce, many cases of glaucoma go undiagnosed or are poorly managed, leading to higher rates of irreversible and avoidable blindness (Varma et al., 2011; Delgado et al., 2019). This paper seeks to understand if a two-pronged community-based strategy, which combined an educational campaign with an intraocular pressure screening, was an effective means to increase ophthalmic care seeking behavior in Roatán, Honduras. To do this, community members were given information about glaucoma and their pressure was taken with an iCare tonometer. Those with elevated pressures were recorded and contacted about follow-up. These data were then compared to the number and identity of patients who sought out ophthalmic care during a one-week medical brigade on the island to determine if the community-based approach was effective in increasing screening and detection of glaucoma. With a 7.56% follow-up rate, the analysis determined that the implemented strategy was ineffective. This study reveals a lack of timely follow-up in Roatán, demonstrating limitations in the utilized method of screening and referral. Additional interventions should be examined to determine more successful methods to increase detection of glaucoma in Roatán
Different Resistance Exercise Loading Paradigms Similarly Affect Skeletal Muscle Gene Expression Patterns of Myostatin-Related Targets and mTORC1 Signaling Markers.
No full textAlthough transcriptome profiling has been used in several resistance training studies, the associated analytical approaches seldom provide in-depth information on individual genes linked to skeletal muscle hypertrophy. Therefore, a secondary analysis was performed herein on a muscle transcriptomic dataset we previously published involving trained college-aged men (n = 11) performing two resistance exercise bouts in a randomized and crossover fashion. The lower-load bout (30 Fail) consisted of 8 sets of lower body exercises to volitional fatigue using 30% one-repetition maximum (1 RM) loads, whereas the higher-load bout (80 Fail) consisted of the same exercises using 80% 1 RM loads. Vastus lateralis muscle biopsies were collected prior to (PRE), 3 h, and 6 h after each exercise bout, and 58 genes associated with skeletal muscle hypertrophy were manually interrogated from our prior microarray data. Select targets were further interrogated for associated protein expression and phosphorylation induced-signaling events. Although none of the 58 gene targets demonstrated significant bout x time interactions, ~57% (32 genes) showed a significant main effect of time from PRE to 3 h (15↑ and 17↓, p p RHEB was the only mTORC1-associated mRNA that was upregulated following exercise. Phosphorylated (phospho-) p70S6K (Thr389) (p = 0.001; PRE to 3 h) and follistatin protein levels (p = 0.021; PRE to 6 h) increased post-exercise, regardless of the bout, whereas phospho-AKT (Thr389), phospho-mTOR (Ser2448), and myostatin protein levels remained unaltered. These data continue to suggest that performing resistance exercise to volitional fatigue, regardless of load selection, elicits similar transient mRNA and signaling responses in skeletal muscle. Moreover, these data provide further evidence that the transcriptional regulation of myostatin signaling is an involved mechanism in response to resistance exercise
Differences in Cognitive Task Performance, Reinforcement Enhancement, and Nicotine Dependence Between Menthol and Nonmenthol Cigarette Smokers.
No full textIntroductionMenthol has been shown to target similar brain regions and neural receptors as nicotine, yet the association between menthol cigarette use and cognitive performance remains unknown.Aims and methodsThis study examined differences in cognitive task performance between menthol (MS) and nonmenthol (NMS) cigarette smokers after acute cigarette consumption. Sixty white and black and/or African American, nonabstinent, MS (n = 30) and NMS (n = 30) were assessed presmoking and postsmoking their preferred cigarette on four computerized tasks: Continuous Performance Task (CPT; alerting attention), N-Back Task (working memory), Finger Tapping Task (motor control), and Apple Picker Task (reinforcement enhancement). Self-reported nicotine dependence and objective smoking topography measures were also compared between groups.ResultsInitial unadjusted analyses showed a significant effect of cigarette type × time on CPT speed (p = .042), where MS improved while NMS group worsened in CPT speed after smoking. After controlling for baseline cigarette craving and cigarette nicotine levels, the effect of cigarette type × time for all cognitive outcomes was statistically nonsignificant (ps > .05). However, there remained a significant effect of cigarette type, where MS versus NMS had poorer CPT (p = .046) and N-Back Task accuracy (p = .006) but faster N-Back speed (p = .039). There were no statistically significant differences between groups on reinforcement enhancement, nicotine dependence, or smoking behavior outcomes (ps > .05).ConclusionsContrary to our hypotheses, results did not find a significant effect of cigarette type on the change in cognitive performance after acute smoking in nonabstinent smokers. Further studies are needed to clarify the specific pharmacological effects of nicotine and menthol on cognitive functioning.ImplicationsThe current study is the first to compare the potential enhancement of cognitive task performance after acute cigarette smoking between satiated menthol and nonmenthol cigarette smokers. Study results suggest that acute menthol cigarette use may not enhance cognitive function above and beyond nonmenthol cigarettes to increase dependence among menthol smokers. However, the contribution of other psychological factors (eg, craving, mood) and cigarette characteristics (eg, nicotine content) may be involved in cognitive function enhancement to perpetuate dependence and smoking persistence for menthol smokers
Perioperative Pain Management for Elective Spine Surgery: Opioid Use and Multimodal Strategies.
No full textIn recent years, physicians and institutions have come to recognize the increasing opioid epidemic in the United States, thus prompting a dramatic shift in opioid prescribing patterns. The lack of well-studied alternative treatment regimens has led to a substantial burden of opioid addiction in the United States. These forces have led to a huge economic burden on the country. The spine surgery population is particularly high risk for uncontrolled perioperative pain, because most patients experience chronic pain preoperatively and many patients continue to experience pain postoperatively. Overall, there is a large incentive to better understand comprehensive multimodal pain management regimens, particularly in the spine surgery patient population. The goal of this review is to explore trends in pain symptoms in spine surgery patients, overview the best practices in pain medications and management, and provide a concise multimodal and behavioral treatment algorithm for pain management, which has since been adopted by a high-volume tertiary academic medical center
Enhanced HIF2α expression during human trophoblast differentiation into syncytiotrophoblast suppresses transcription of placental growth factor.
No full textPlacental growth factor (PlGF), abundantly produced from trophoblasts is involved in placental angiogenesis. The regulatory mechanism of its expression is poorly understood. Hypoxia inducible factors (HIFs) are centrally involved in the modulation of cellular function in response to low oxygen conditions. This study aimed to clarify HIF1α and HIF2α expression patterns during cytotrophoblast differentiation into syncytiotrophoblast and the impact of any changes on PlGF expression. HIF proteins were induced remarkably under low oxygen condition (2%). HIF1α expression decreased and HIF2α expression increased when syncytialization of cultured cytotrophoblasts is progressed. Those expression changes of HIF proteins in the process of in-vitro syncytialization was congruent with the immunohistochemical findings in preeclamptic placenta as well as uncomplicated placenta. Low oxygen condition was also associated with reduced PlGF production in syncytializing primary cells and BeWo choriocarcinoma cells. Small interfering RNA-mediated HIF2α knockdown in BeWo cells abrogated hypoxia-associated decreases in PlGF secretion; HIF1α silencing had no significant effect on PlGF secretion. In summary, HIF2α, rather than HIF1α, is most affected by reduced oxygen level during syncytialization and increases in HIF2α trigger a reduction of PlGF production. Our findings suggest new and important connections between HIF proteins and PlGF pathways in the regulation of placental angiogenesis
EBV-Associated Gastric Cancer: From Initial Infection to Unique Therapeutic Approaches
No full textEpstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects over 95% of the adult population. While infection is typically asymptomatic, in some individuals, particularly the immunocompromised, EBV is the causative agent of several cancers including lymphomas and epithelial cancers. Specifically, gastric carcinoma, nasopharyngeal carcinoma, and lymphoepithelioma-like carcinoma which occurs across multiple sites in the body, most notably, in the lung. EBV-associated gastric cancer (EBVaGC) is a unique subset of gastric cancer that makes up 10% of all GCs worldwide. EBVaGCs display an 80% rate of activating PIK3CA mutation and are also the most hypermethylated of any tumor type, displaying what is known as a CpG island hypermethylator phenotype (CIMP). EBV infection of B cells can be easily modeled in vitro using primary B cells and lymphoblastoid cell lines (LCLs). However, epithelial cell infection models have proven much more difficult to develop. Given these difficulties, EBV infection and outgrowth in epithelial cells is comparatively understudied and the process of tumorigenesis in vivo is poorly understood. In this dissertation, I developed methods to generate EBV infected epithelial cell lines derived from both gastric cancer and lung adenocarcinoma using diverse strains of EBV. I used these models to complete a CRISPR/Cas9 whole genome knockout screen to identify cellular restriction factors of infection and outgrowth. Together, these data will provide novel insights into the process of EBV infection and the dynamic interplay between virus and host during tumorigenesis. Furthermore, I have used these models to explore unique therapeutic approaches for EBV+ epithelial cancers. Specifically, I have characterized the lytic reactivation potential to histone deacetylase (HDAC) inhibitors and generated preclinical data supporting the use of HDAC inhibitors and the anti-viral ganciclovir for treatment of EBV+ epithelial tumors. Lastly, I have identified modulators of the response to a PI3Ka inhibitor in PIK3CA mutant gastric cancers. I found that loss of NEDD9 or inhibition of BCL-XL rendered cells hyper-sensitive to the PI3Ka inhibitor BYL719. Additionally, I found that loss of CBFB conferred resistance to BYL719 through up- regulation of the protein kinase PIM1 and defined the clinical utility of our data in the context of PI3K inhibition more broadly. The work outlined in this dissertation contributes to the study of EBV infection and tumorigenesis in the stomach as well as provides mechanistic insights into novel therapeutic approaches for EBV+ epithelial cancers and PIK3CA mutant gastric cancers.</p
Nuclear PTEN Regulates Thymidylate Biosynthesis and Cellular Sensitivity to Antifolate Treatment
No full textMetabolic reprogramming contributes to tumorigenesis and holds significant promise for cancer therapy. The PTEN tumor suppressor governs a variety of biological processes, including metabolism, by acting on distinct molecular targets in different subcellular compartments. In the cytoplasm, PTEN regulates a plethora of metabolic processes through antagonizing the PI3K/AKT/mTORC1 pathway. However, the metabolic regulation of PTEN in the nucleus remain undefined. Using a gain-of-function approach to examine the metabolic consequences of PTEN targeted to different sub-cellular compartments in human prostate cancer cell lines, we reveal a nuclear function for PTEN in controlling de novo thymidylate biosynthesis and may also open novel therapeutic avenues for targeting nuclear-excluded PTEN prostate cancer cells with anti-folate cancer treatment. The first four chapters of this dissertation are introductory information that outlines the role of PTEN in cancer, the importance of metabolic compartmentalization, the fundamentals of pyrimidine biosynthesis pathways, and the novelty of anti-folate cancer treatments. Chapter 1 explains the role of PTEN as a tumor suppressor and continues on to discuss its role at the cell membrane as a metabolic regulator. The gap in knowledge in the field is understanding the role nuclear PTEN plays as a metabolic regulator. This is important as nuclear PTEN has been shown to be associated with more aggressive cancer phenotypes. Chapter 2 focuses on the background of metabolic compartmentalization and how, by strategically placing genes and metabolites spatiotemporally, the cell is able to execute key molecular mechanisms more efficiently. In this chapter, we highlight where the current field is with metabolic compartmentalization, the advantages of compartmentalization and how utilization of this knowledge can be used for definitive therapeutics. Chapter 3 focuses on pyrimidine biosynthesis and thymidylate biosynthesis. Thymidylate is synthesized de novo by thymidylate synthase (TYMS), with the enzymes dihydrofolate reductase (DHFR) and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) or serine hydroxymethyltransferase (SHMT) which are required to regenerate 5, 10-methylenetetrahydrofolate. MTHFD1 is the primary source of 5,10-methylenetetrhydrofolate generation, and therefore its proper function in the nucleus ensures the functioning of de novo thymidylate biosynthesis. Using Mass-Spectrometry, we discovered that MTHFD1 is a top candidate protein interacting with PTEN in human prostate cancer cells. This is the key focus of this paper and will be important background for Chapter 7 and 8 which delve into the thymidylate pathway and the potential role of nuclear PTEN. A deeper understanding of nuclear PTEN’s regulation of MTHFD1 may, in turn, open new therapeutic avenues for anti-folate cancer treatment tailored to PTEN sub-cellular localization. This brings us to Chapter 4, which focuses on the current market of anti-folate treatment and the importance of precision medicine and combinatorial treatment.
Chapter 5 is the start of our project, it is the basis of the remainder chapters and what allowed us to elucidate the importance of nuclear PTEN. To explore the role of PTEN as not just a tumor suppressor, but as a metabolic regulator, we first sought to generate overexpression cell lines with PTEN localized to various subcellular compartments. As explained in chapter 1 and 2, we discuss how in the cytosol inactive PTEN can be recruited to the plasma membrane where it functions as a lipid phosphatase to suppress the activation of the proto-oncogenic phosphoinositide 3-kinase (PI3K)–AKT-mTOR signaling pathway. In the nucleus, PTEN acts to induce cell cycle arrest and maintain genomic stability. However, the role of PTEN in metabolism is incompletely understood. It is clearly understood that each subcellular compartment harbors specific metabolic activities and PTEN is present in different subcellular locations where it performs distinct functions acting on specific effectors. To explore this, we used a gain-of-function approach to examine the metabolic consequences of PTEN targeted to different sub-cellular compartments. Plasmids were generated using site-directed mutagenesis and PCR. We used the vector pTRIPZ, which is an inducible TET-ON system. This was necessary as overexpression of a tumor suppressor in cancer cell lines, if left permanently on, leads to slow cell growth and expulsion of the plasmid by inherent cancer cell mechanisms. We generated a vector plasmid, which was used as the baseline for all experiments, a wildtype PTEN plasmid, which was “normal” PTEN and PTEN had the ability to localize to the membrane or nucleus as it pleased, a cytoplasmic membrane plasmid, which localized PTEN permanently to the membrane, nuclear PTEN, which localized PTEN permanently to the nucleus, and mutant C124S PTEN, which generates a catalytically silent PTEN variant preventing its role at the membrane and its involvement in the PI3K-AKT-mTOR pathway. All of the plasmids were transfected as a lentivirus into PC3 and C4-2 prostate cancer cells. Both cell lines are PTEN-null and C4-2 represents early stage prostate cancer and is AR positive, while PC3 cells represent later stage metastatic prostate cancer and are AR negative. Prostate cancer is one of the leading causes of morbidity and mortality in the world. Identification of novel therapeutics to combat this deadly disease is still needed today, despite advances in PSA testing, molecular diagnostics, and androgen-deprivation therapy treatments. Inactivation of PTEN by deletion or mutation is found in 20% of primary prostate tumor samples and over 50% of castration-resistant tumors. This highlights the importance of using prostate cancer cell lines as a model system. Cell lines were confirmed for PTEN localization by 3 methods: Western blotting, subcellular fractionation assays, and immunofluorescence assays. Confirmation of all 5 cell lines in both PC3 and C4-2 systems sets up the system used in the next couple chapters.
Chapter 6 focuses on the metabolic profiling of our cell lines and highlights the big data generated opening up multiple avenues to be explored. It emphasizes the potential role of PTEN as not only a tumor suppressor, but as a metabolic regulator. Unbiased metabolic profiling was performed on PC3 and C4-2 overexpression cell lines. A polar metabolomics profiling platform using selected reaction monitoring with the 5500 QTRAP hybrid triple quadrupole mass spectrometer, created by Dr John Asara, was utilized for metabolomics data generation. Using LC MS/MS for polar metabolite profiling the platform is a single normal phase hydrophilic interaction liquid chromatographic run (HILIC) and has a short mass spectra acquisition time of only 15 minutes. The platform allows for over 250 metabolic compounds to be targeted without chromatographically scheduled selected reaction monitoring. Lastly, all analysis of data is done through Metaboanalyst, and output files are generated in R command. Large data sets were generated for each cell type and allowed for multiple avenues of research for this dissertation. Looking at a heat map of all 5 cell lines, it was clear nuclear PTEN stood out and followed a different and opposite pattern to that of wildtype and cytoplasmic membrane PTEN. This led us to compare nuclear PTEN to vector PTEN null cell lines and determine which pathways were enriched. Pyrimidine biosynthesis was at the top of the list, which leads us to chapter 7.
Chapter 7 focuses on our analysis of pyrimidine biosynthesis and the potential role nuclear PTEN plays in it. The absence of nuclear PTEN is associate with more aggressive disease in patients and therefore can serve as a useful biomarker and potential therapeutic avenue. While purine metabolism happens exclusively in the cytoplasm, pyrimidine metabolism occurs in the cytoplasm, mitochondria, and nucleus. Most notable from our data, in relation to pyrimidine pathways, was the upregulation of dTMP levels. dTMP, as mentioned in chapter 3, is produced from dUMP and both metabolites participate in thymidine biosynthesis. Nuclear localization of the dTMP biosynthesis pathway is a critical factor for allowing the pathway to play its role in DNA synthesis and proper cell division. This pathway utilizes folate one-carbon metabolism of which the enzyme MTHFD1 is an important facilitator of the pathway. Previous data shows, through mass spectrometric analysis, that MTHFD1 was identified as a PTEN interacting protein. This information suggests a potential role that PTEN plays in thymidylate biosynthesis. This chapter aims to highlight the potential roles PTEN plays in thymidylate biosynthesis and pave the way for novel therapeutic treatment.
Chapter 8 discusses the findings in chapter 7 and their ability to contribute to the metabolic therapeutic field. We utilized crystal violet assays to measure cell growth over a 6 day time period and treated cells with both an anti-folate and anti-PI3K drug to maximize efficacy against nuclear PTEN-null cancer patients. Pharmaceuticals that target enzymes in folate-dependent dTMP biosynthesis are developed as anti-cancer drugs. 5-fluorouracil (5-FU) is the most widely used anti-folate cancer drug and focuses on inhibition of TYMS. A full discussion of 5-FU’s pathway is discussed in chapter 4. The information and literature lead us to hypothesize that presence of nuclear-PTEN would affect 5-FU efficacy and combinatorial therapeutics would be a novel treatment method. In combination with 5-FU we used Buparlisib, an oral PI3K inhibitor, to inhibit tumor growth via the PI3K pathway which has been shown to promote castration and chemotherapy resistance. We sought to determine if BKM used in combination with 5-FU could be a potential therapeutic option for patients with nuclear PTEN loss. Previous data shows evidence of a protective effect of nuclear PTEN on drug treatment suggesting novel precision therapeutic treatment for loss of nuclear PTEN patients.
Chapter 9 concludes our findings and provides combinatorial therapeutic pathways for metastatic cancer cell lines that have PTEN deletions and or mutations. The chapter also explores future directions for the work, highlights the contributions to the field of PTEN and metabolism, and recognizes PTEN as a metabolic regulator and gene that is vital in cancer tumorigenesis progression, applicable pan-cancer.
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