Browsing by Subject "Health Sciences, Oncology"

Normal cells require input from extrinsic growth factors to control proliferation and survival. Recent studies have demonstrated that these same extrinsic signals also regulate cellular metabolism to ensure that metabolism adequately supports the demands of cell function, proliferation, and cell survival. The PI3K/Akt pathway is downstream of many growth factors and can promote both glucose metabolism and cell survival. Aberrant activation of the PI3K/Akt pathway is common in cancer, and its activation can contribute to the growth factor independence that is a hallmark of neoplastic cells. Metabolic demand is high in stimulated and leukemic T cells, and activation of Akt can increase glucose metabolism to meet these requirements. There is great interest in targeting the unique metabolism of cancer cells for cancer therapy, thus making an understanding of the interaction of metabolism and cell death essential.

Akt is also anti-apoptotic and can inhibit cell death by regulating members of the Bcl-2 family. Interestingly, the ability of Akt to prevent cell death is inextricably linked to its metabolic function. Several recent studies have demonstrated that glucose metabolism can affect Bcl-2 to family members to promote cell survival, but the role of Akt-dependent glucose metabolism in the regulation of Bcl-2 family members is not understood. Using a model of growth factor withdrawal-induced apoptosis, we show that Akt prevents cell death by maintaining glucose metabolism to regulate the Bcl-2 family members Puma and Mcl-1, and demonstrate the importance of this pathway in the survival of stimulated T lymphocytes and leukemia.

After growth factor withdrawal, active Akt suppressed Puma induction in abundant glucose, but Puma was rapidly upregulated in glucose-deficient conditions and was necessary and sufficient to promote efficient cell death. Importantly, glucose was not uniquely required, as provision of alternative mitochondrial fuels allowed Akt to suppress Puma and maintain survival. This metabolic regulation of Puma was mediated through partially p53-dependent transcriptional induction as well as control of Puma protein stability.

In addition to inhibiting Puma expression, active Akt prevented the loss of Mcl-1 after growth factor withdrawal by sustaining Mcl-1 protein synthesis in a glucose-dependent manner. Mcl-1 was essential for preventing Bim-induced apoptosis, as Akt could not inhibit Bim induction after growth factor deprivation. Slowing of Mcl-1 synthesis by inhibiting glucose metabolism reversed Mcl-1-mediated resistance of leukemic cells to the Bcl-2 inhibitor ABT-737. Importantly, Akt and glucose-reliant Mcl-1 expression required mTOR-dependent phosphorylation of 4EBP, and treatment with mTOR inhibitors also reversed ABT-737 resistance.

Together, this study demonstrates that Akt promotes cell survival by preventing metabolic checkpoints that stimulate Puma expression and stability and inhibit Mcl-1 synthesis, advancing our understanding of the links between metabolism and cell death. These studies highlight the importance of cellular metabolism--including a potential role for the alternative sugar fructose--in cancer cell survival that may provide a mechanistic understanding to drive development of metabolism-targeted cancer therapies.

Tumor hypoxia is an enduring problem for traditional cancer therapies such as radiation and chemotherapy. This obstacle has traditionally been attributed to the widespread presence of chronic, diffusion-limited hypoxia in solid tumors; recent data suggests that tumor hypoxia may also be spatially and temporally variable. In this work we characterize the presence of spatial and temporal fluctuations in hypoxia, as well as use mathematical modeling to predict the impact of fluctuations on the hypoxic cytotoxin, tirapazamine, and examine potential mechanisms of fluctuations in tumor oxygenation. Using phosphorescence lifetime imaging on preclinical tumors, we show that instabilities in tumor oxygenation are a prevalent characteristic of three tumor lines and that previous characterization of tumor hypoxia as being primarily diffusion-limited does not accurately portray the tumor microenvironment. Then, using a one-dimensional theoretical model, we examine the effects of fluctuating hypoxia on metabolized tirapazamine concentration; we find that fluctuating oxygen reduces the concentration of metabolized tirapazamine at distances farther from the source, thereby limiting its ability to reach and kill the most hypoxic cells. Finally, we use a three-dimensional Green's function oxygen transport model to explore the effects of temporal fluctuations in hemoglobin saturation, blood flow, and overall perfusion on tumor tissue oxygenation. Results from the model predict that hemoglobin saturation has a dominant effect on tissue oxygenation. These studies collectively suggest that the pervasive temporal and spatial heterogeneity in tumor oxygenation are highly therapeutically relevant, and future clinical and preclinical studies are needed.

Accurate delivery of external beam radiation therapy relies on localization of the treatment target. For this work, the accuracy of an electromagnetic localization system (ELS) was first verified. Next, prostate deformations and rotations occurring throughout therapy were analyzed. Motion studies were also conducted to investigate the use of the ELS during intensity modulated arc therapy (IMAT) for prostate cancer. Lastly, appropriate margins were determined and new plans utilizing smaller margins were tested for two patients who exhibited large transponder displacements. Electromagnetic alignments were accurate to within 1mm as compared to x-ray imaging. All patients fell within the default geometric residual limit (2mm) and most fell within the default rotation limit (10°). The ELS appeared to be suitable for use during IMAT with a 5mm margin. A 3mm margin was tested and was adequate when the main displacements were translational shifts; however, it was not adequate when large rotational displacements occurred.

We present a new x-ray imaging technique, Correlation Imaging (CI), for improved breast and lung cancer detection. In CI, multiple low-dose radiographic images are acquired along a limited angular arc. Information from unreconstructed angular projections is directly combined to reduce the effect of overlying anatomy - the largest bottleneck in diagnosing cancer with projection imaging. In addition, CI avoids reconstruction artifacts that otherwise limit the performance of tomosynthesis. This work involved assessing the feasibility of the CI technique, its optimization, and its implementation for breast and chest imaging.

First a theoretical model was developed to determine the diagnostic information content of projection images using a mathematical observer. The model was benchmarked for a specific application in assessing the impact of reduced dose in mammography. Using this model, a multi-factorial task-based framework was developed to optimize the image acquisition of CI using existing low-dose clinical data. The framework was further validated using a CADe processor. Performance of CI was evaluated on mastectomy specimens at clinically relevant doses and further compared to tomosynthesis. Finally, leveraging on the expected improvement in breast imaging, a new hardware capable of CI acquisition for chest imaging was designed, prototyped, evaluated, and experimentally validated.

The theoretical model successfully predicted diagnostic performance on mammographic backgrounds, indicating a possible reduction in mammography dose by as much as 50% without adversely affecting lesion detection. Application of this model on low-dose clinical data showed that peak CI performance may be obtained with 15-17 projections. CAD results confirmed similar trends. Mastectomy specimen results at higher dose revealed that the performance of optimized breast CI may exceed that of mammography and tomosynthesis by 18% and 8%, respectively. Furthermore, for both CI and tomosynthesis, highest dose setting and maximum angular span with an angular separation of 2.75o was found to be optimum, indicating a threshold in the number of projections per angular span for optimum performance.

Finally, for the CI chest imaging system, the positional errors were found to be within 1% and motion blur to have minimal impact on the system MTF. The clinical images had excellent diagnostic quality for potentially improved lung cancer detection. The system was found to be robust and scalable to enable advanced applications for chest radiography, including novel tomosynthesis trajectories and stereoscopic imaging.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.

Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions.

The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.

The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.

Purpose: Changes in patient volume, due to tumor shrinkage, dehydration, dysphagia and atrophy, could present issues in the accuracy of dosimetry throughout the course of treatment. The aim of this work is to study the dosimetric impacts of the volumetric changes during IMRT and to investigate the feasibilities of electronic portal imaging device (EPID) in predicting the impacts. Materials and Methods: An anthropomorphic head and neck phantom was used to represent two scenarios: symmetric and asymmetric volume loss. The phantom was simulated and planned according to the head and neck protocols used in our clinic. Dose volume histograms (DVH) were generated for each set up scenario and were used to calculate the integral dose expected at the coincident volume of the phantom. During treatment delivery, the EPID captured exit fluence of each beam at each level of bolus thickness. These images were quantitatively analyzed using gamma analysis with criteria of 3% and 3mm dose difference and distance-to-agreement respectively. Results: Comparing maximum to minimum volume in the symmetric situation with DVH generated in Eclipse show substantial fluctuations in dose. When comparing five layers of bolus material to zero layers of bolus material, the changes were most significant. The asymmetric volume change predicted dose fluctuations that were less significant than the symmetric phantom. As for gamma analysis, a quantitative evaluation of the integrated dose fluence, captured by the EPID, showed extreme variability in the images with five layers of bolus when compared to images with no bolus. Less significant variation was shown in layers of closer thicknesses, as expected. Conclusions: The phantom study indicates that volume loss could contribute to clinically considerable changes in the dose delivered to target and organs at risk. The proposed technique using EPID could provide valuable information about the variation of dose due to volumetric changes and might be potentially useful.

Selective therapeutic targeting of tumors requires identification of differences between the homeostatic requirements of cancer and host cells. One such difference is the manner in which cancer cells acquire energy. Cancer cells often grow in an environment of local hypoxia; under these conditions tumor cells depend on glycolysis for energy, but are unable to perform oxidative phosphorylation. Many tumor cells, despite normoxic conditions, continue to perform glycolysis without oxidative phosphorylation. The net result of glycolysis without oxidative phosphorylation is twofold: the need to consume a greater amount of glucose than a non-cancerous host cell, and the burden of increased intracellular lactic acid. The proteins responsible for the transport of lactic acid in and out of cells are known as the monocarboxylate transporters (MCTs). Monocarboxylate Transporter 1 (MCT1) and Monocarboxylate Transporter 4 (MCT4) are the MCTs that play a major role in the transport of lactic acid. Tumor cells depend on MCT1 and MCT4 activity to excrete excess intracellular lactic acid to maintain neutral intracellular pH and homeostasis. Using human neuroblastoma and prostate cancer cell lines this work demonstrates that tumor cells can be selectively targeted tumor under conditions of hypoxia or acidosis in vitro with the drug lonidamine, with a small molecule inhibitor selective for MCT1, or with RNA interference of MCT1. Inhibition of MCT1 activity in neuroblastoma cells under acidic extracellular conditions results in intracellular acidification and cell death. MCT1 mRNA is expressed in human neuroblastoma and positively correlated with clinical risk profile. Inhibition of MCT1 activity in hypoxic prostate cancer cells results in a reduction of lactate excretion, decreased intracellular pH, inhibition of ATP production, and subsequent cell death. MCT1 expression in sections of human prostate tumors has been demonstrated to validate MCT1 as a target in prostate cancer.

Through the Pasteur and Warburg effects, tumors have an increased demand for glucose. Some cancers store glycogen, but the reasons for this are largely unknown. It is hypothesized that tumor glycogen is used to promote tumor survival during transient hypoxia or low glucose, and that the mechanisms by which glycogen is stored is a potential therapeutic target in cancer. Tumors from human cell lines (WiDr, PC3, FaDu) have been grown in nude mice, sectioned and stained to measure glycogen storage. Using consecutive frozen sections, levels of hypoxia, glucose, lactate, ATP, and CD31, an endothelial cell marker, have been determined. These sections have been employed to elucidate the "architecture" of tumor metabolism in terms of vessel distance. Additionally, PAS-stained EF5 labeled human tumor samples were used to obtain calibrated hypoxia measurements to correlate with PAS. These studies demonstrate a correlation between hypoxia and the formation of glycogen deposits in human tumors and nude mouse xenografts. In cell culture, formation of glycogen deposits after exposure to hypoxia has been demonstrated, in addition to expression of glycogen synthase in human cancer cell lines.

The development of novel selective cancer chemotherapeutics will require the identification of differences between cancerous cells and normal host cells to exploit as targets. Several differences in metabolism, including the need to excrete excess lactic acid and store glycogen under hypoxic conditions, are such targets. Novel therapeutics exploiting these targets should be effective against cancer cells and minimally toxic to host cells.

On-board imaging is useful for guiding radiation to patients in the treatment position; however, current treatment-room imaging modalities are not sensitive to physiology - features that may differentiate tumor from nearby tissue or identify biological targets, e.g., hypoxia, high tumor burden, or increased proliferation. Single photon emission computed tomography (SPECT) is sensitive to physiology. We propose on-board SPECT for biological target localization.

Localization performance was studied in computer-simulated and scanner-acquired parallel-hole SPECT images. Numerical observers were forced to localize hot targets in limited search volumes that account for uncertainties common to radiation therapy delivery. Localization performance was studied for spherical targets of various diameters, activity ratios, and anatomical locations. Also investigated were the effects of detector response function compensation (DRC) and observer normalization on target localization. Localization performance was optimized as a function of iteration number and degree of post-reconstruction smoothing. Localization error patterns were analyzed for directional dependencies and were related to the detector trajectory. Localization performance and the effect of the detector trajectory were investigated in a hardware study using a whole-body phantom.

Typically targets of 6:1 activity were localized as accurately using 4-minute scans as those of 3:1 activity using 20-minute scans. This trend is consistent with the relationship between contrast and noise in the contrast-to-noise ratio (CNR) and implies that higher contrast targets are better candidates for on-board SPECT because of time constraints in the treatment room. Using 4-minute scans, mean localization errors were within 2 mm for superficial targets of 6:1 activity that were proximal to the detector trajectory and of at least 14 mm in diameter. Localization was significantly better (p < 0.05, Wilcoxon signed-rank test) with than without observer normalization and DRC at 5 of 6 superficial tumor sites. Observer normalization improved localization substantially for a target proximal to the much hotter heart. Localization error patterns were shown to be anisotropic and dependent on target position relative to the detector trajectory. Detector views of close approach and of minimal attenuation were predictive of directions with the smallest (magnitude) localization bias and precision. The detector trajectory had a substantial effect on localization performance. In scanner-acquired SPECT images, mean localization errors of a 22-mm-diameter superficial target were 0.8, 1.5, and 6.9 mm respectively using proximal 180°, 360°, and distal 180° detector trajectories, thus demonstrating the benefits of using a proximal 180° detector trajectory.

In conclusion, the potential performance characteristics of on-board SPECT were investigated using computer-simulation and real-detector studies. Mean localization errors < 2 mm were obtained for proximal, superficial targets with diameters >14 mm and of 6:1 activity relative to background using scan times of approximately 5 minutes. The observed direction-dependent localization errors are related to the detector trajectory and have important implications for radiation therapy. This works shows that parallel-hole SPECT could be useful for localizing certain biological targets.

The role of two cell extrinsic signals, T cell receptor (TCR) ligation and interleukin-7, in promoting glucose uptake and survival of T lymphocytes is examined in this work. Both of these signals are capable of regulating the uptake and fate of glucose, but the requirement of this regulation for T cell homeostasis and functionality remains unclear. To examine the role of TCR mediated increases of glucose metabolism and the signals involved, primary murine T cells were activated in vitro and the role and regulation of glucose uptake was examined. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required for maximal glucose uptake following TCR stimulation by upregulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active Akt (mAkt) increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent upregulation of Glut1 protein and Akt-dependent Glut1 cell surface trafficking. In support of this model, co-expression of Glut1 and mAkt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism necessary for T cell activation and proliferation.

Glucose uptake is dramatically increased in response to TCR and costimulation signaling, however, glucose uptake must be maintained at a low level in naive T cells to promote survival and homeostasis. Interleukin-7 (IL-7) plays a central role in maintaining naive T cell homeostasis, and mediates this effect in vivo at least in part through control of homeostatic proliferation and inhibition of apoptosis. IL-7 can promote glucose uptake and glycolysis in vitro and may also promote glucose metabolism in vivo to maintain T cell survival. To determine if IL-7 regulates T cell metabolism in vivo, we generated a transgenic model for conditional IL-7 receptor (IL 7R) expression on IL-7R-/- T cells. T cells in this model developed normally and, consistent with previous work, deletion of the IL-7R transgene in vivo led to cell death even in an otherwise normal lymphoid compartment. Importantly, in vivo deletion of IL 7R also led to decreased cell size and glycolytic flux. However, glucose uptake was not altered following deletion of the IL-7R indicating that while not essential for glucose uptake, IL-7 is required for maintenance of glycolysis. These data are the first to identify a signal required in vivo to regulate lymphocyte metabolism and demonstrate that in addition to its well-defined roles in homeostatic proliferation and cell survival, IL-7 plays a key and non-redundant role to maintain T cell glycolysis. Together, these data concerning the role of TCR, costimulation, and IL-7 in the regulation of glucose uptake and metabolism exemplify the importance of cell extrinsic signals and the regulation of glucose utilization.

MAL (myelin and lymphocyte protein), has been implicated in several malignancies including esophageal, gastric, and cervical cancers. We have demonstrated that the MAL protein is expressed in the normal breast epithelium, and aberrantly expressed in breast cancer. Bisulfite sequencing of the MAL promoter CpG island revealed hypermethylation in breast cancer cell lines and 69% of primary tumors analyzed compared with normal breast epithelial cells. Differential methylation between normal and cancer DNA was confined to the proximal promoter region. In a subset of breast cancer cell lines, promoter methylation correlated with transcriptional silencing that was reversible with the methylation inhibitor decitabine. Furthermore, exogenous expression of MAL in breast cancer cell lines resulted in decreased cell proliferation, motility, reduced cell invasion through Matrigel and suppressed anchorage-independent growth in soft agar. In a cohort of 122 primary breast tumors, immunohistochemical analysis revealed that the MAL protein was an independent predictor of benefit from adjuvant chemotherapy. Moreover, overexpression of MAL in triple-negative MDA-MB-468 and BT20 breast cancer cell lines was sufficient to confer sensitivity to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibition and was associated with reduced phosphatidylinositol-3 kinase (PI3K)/Akt signaling. Immunohistochemistry studies conducted on 144 late-stage serous ovarian cancers showed that MAL expression was a significant predictor of survival. Knockdown of MAL expression in the SKOV8 ovarian cancer cell line reduced cell proliferation and resulted in increased sensitivity to the chemotherapeutic drug carboplatin. Thus, we have identified the MAL gene as a novel epigenetically regulated gene in breast cancer with implications for response to chemotherapy in both breast and ovarian cancer. Furthermore, we have shown that the MAL protein has predictive and prognostic value in breast and ovarian cancers, respectively.

Signaling from the G protein, Rap1 is involved in several fundamental biological processes. Yet the mechanism or even consequence of Rap1 signaling in several biologies and diseases is still unclear. Rap1 has been implicated in cancer tumorigenesis, but its role in cancer invasion and metastasis is less understood. Rap1 signals to pathways involved in cell adhesion, migration, and survival, suggesting that Rap1 may promote several processes associated with metastasis. Recent studies in another biological system have demonstrated that the Rap activator proteins, Epac, are important regulators of pancreatic β-cell insulin secretion. However, the role of Rap1 in β-cell biology has not yet been defined. Here we established roles for Rap1 in distinct signaling events and begin to answer some of the key questions about Rap1 function in two diverse biologies: cancer metastasis and pancreatic islet β-cell function.

Elucidating the mechanisms of prostate and breast cancer survival and metastasis are critical to the discovery of novel therapeutic targets. Examination of prostate cancer cell lines revealed cells with a high metastatic ability exhibited increased Rap1 activity and reduced expression of the negative regulator, Rap1GAP. Activation of Rap1 increased prostate and breast cancer cell migration and invasion, and inhibition of Rap1A activity via RNAi-mediated knockdown or ectopic expression of Rap1GAP markedly impaired cancer cell migration and invasion. Additional studies implicated integrins α4, β3, and αvβ3 in the mechanism of Rap1-mediated prostate and breast cancer migration. Furthermore, these same integrins and matrix metalloproteinases were shown to be involved in Rap1-induced prostate cancer invasion. Introduction of activated Rap1 into prostate cancer cells dramatically enhanced the rate and incidence of CaP metastasis in a mouse metastasis model. In another mouse xenograft model, blockade of Rap1 signaling by expression of Rap1GAP abrogated breast cancer metastastasis. These studies support a role for aberrant Rap1 activation in prostate and breast cancer metastatic progression, and suggest that targeting Rap1 signaling could provide a means to control metastatisis of these cancers.

In a seperate biological system, the effects of Rap1 signaling on pancreatic β-cells was directly examined. Activation of Rap1 was demonstrated to promote ribosomal protein S6 phosphorylation through the mTOR and p70 S6 kinase (S6K1) pathway, a known growth-regulatory pathway. This newly defined β-cell axis acts downstream of cAMP, in parallel with the stimulation of both Epac and PKA. Like previous studies on Epac, activation of Rap1 indeed increased glucose stimulated insulin secretion (GSIS) from rat islet β-cells; however, Rap1-mediated GSIS did not appear to signal through this new S6 pathway. Interestingly, Rap1 was show to significantly increase islet cell proliferation and this indeed occured through signaling to mTOR and S6. In summary, these findings represent a new link between cAMP signaling and the pathways controlling β-cell proliferation, and suggest that directly targeting this pathway may have beneficial therapeutic effects for patients with Type 2 diabetes. Furthermore, an additional benefit to targeting Rap1 signaling is the potentiation of insulin secretion, which could possibly prevent or reverse β-cell dysfunction (i.e., defects in both β-cell mass and insulin secretory capacity) in diabetes.