Browsing by Subject "Breast-Cancer"

Based on a biopsychosocial model of mammography pain, the current study assessed if specific biological and psychosocial factors were associated with higher reported mammography pain in early stage breast cancer survivors. One hundred and twenty-seven women completed questionnaires assessing demographic information, cancer treatment history, ongoing breast pain, mammography-related anxiety, and social support immediately prior to receiving a mammogram. They then completed questionnaires assessing mammography pain and mammography-related pain catastrophizing immediately following the mammogram. Using path modeling and mediation analyses, relations among these variables were examined. Results revealed that mammography-related pain catastrophizing was related to higher mammography pain directly, while ongoing breast pain, lower social support quantity, and lower perceived quality of social support related to higher mammography pain indirectly through mammography-related pain catastrophizing. Moderated mediation analyses found that the mediation effects of mammography-related pain catastrophizing were significantly different at varying levels of perceived quality of social support, with more pronounced negative effects for those with higher quality support than those with lower quality support. The theoretical, clinical, and research implications of these findings are discussed.

During the course of breast cancer progression, normally dormant tumor-promoting effects of transforming growth factor-beta (TGF-beta) including migration, invasion, and metastasis are unmasked. Although this switch or gain of TGF-beta function has been modeled extensively in in-vivo and in-vitro breast cancer systems, the signaling mechanisms that control this TGF-beta switch are poorly understood. Indeed, the precise role of canonical TGF-beta signaling through the type I TGF-beta receptor, ALK5, and its intracellular effectors, Smad2 and Smad3, is still poorly understood. In an effort to identify mechanisms that regulate the ability of TGF-beta to stimulate mammary epithelial cell migration in-vitro, we found that TGF-beta stimulates the phosphorylation of Smad1 and Smad5, intracellular effectors that are typically associated with bone morphogenetic protein (BMP) signaling. As this phosphorylation response has not been reported extensively, little is known about the prevalance, mechanism, function, or pathological relevance of TGF-beta-stimulated Smad1/5 phosphorylation.

Herein, we use pharmacologic inhibition, RNA interference, and additional biochemical and cell-based approaches to identify a novel mechanism and function for non-canonical TGF-beta signaling through an ALK5-Smad1/5 axis. We show that TGF-beta stimulates Smad1/5 phosphorylation in an ALK5 dependent manner in cells of epithelial, endothelial, and embryonic origin. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of ALK5. Functionally, this phosphorylation event is essential to the initiation and promotion of TGF-beta-stimulated migration in mammary epithelial cells. Interestingly, this phosphorylation event may promote migration by regulating TGF-beta target gene expression, as evidenced by the identification of putative Smad1/5-dependent TGF-beta target genes using microarray analysis. Finally, of particular relevance to mammary tumor progression, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or HER2 oncogene activation.

Taken together, our data provides evidence that TGF-beta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGF-beta switch in mammary epithelial cells.

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.

This thesis research builds upon prior work that developed separate SPECT and CT (computed mammotomography, or breast CT) devices that were independently capable of imaging an uncompressed breast in 3D space. To further develop the system as a clinically viable device, it was necessary to integrate the separate imaging systems onto a single gantry, and to simultaneously design a patient-friendly bed that could routinely and effectively position the patient during dual-modality imaging of her uncompressed breast in the system's common field of view. This thesis describes this process and also investigates practical challenges associated with dedicated breast imaging of a prone patient using the integrated SPECT-CT device.

We initially characterized the practicability of implementing the novel x-ray beam ultra-thick K-edge filtration scheme designed for routine use with the breast CT system. Extensive computer simulations and physical measurements were performed to characterize the x-ray beam produced using K-edge filtration with cerium and to compare it to beams produced using other filtration methods and materials. The advantages of using this heavily filtered x-ray beam for uncompressed breast CT imaging were then further evaluated by measuring the dose absorbed by an uncompressed cadaver breast during the course of a routine tomographic scan. It was found that the breast CT device is indeed capable of imaging uncompressed breasts at dose levels below that of the maximum utilized for dual-view screening mammography.

To prepare the separate SPECT and CT systems for integration onto a single platform, the cross contamination of the image of one modality by primary and scattered photons of the complementary modality was quantified. It was found that contamination levels of the emission (SPECT) image by the x-ray transmission source were generally far less than 2% when using photopeak energy windows up to ±8%. In addition, while there was some quantifiable evidence of a variation in the transmission image in response to the presence of 99mTc photons in the patient, the effect of primary and scattered 99mTc photons on the visibility of 5 mm acrylic photons in a low contrast x-ray transmission environment was negligible.

A novel, tiered, stainless steel patient bed was then designed to allow dual-modality imaging using the integrated SPECT-CT system. The performance of the hybrid SPECT-CT system was evaluated during early stage dual-modality patient imaging trials with particular emphasis placed on the performance of the patient bed. The bed was successful in its primary task of enabling dual-modality imaging of a patient's breast in the common field of view, but practical challenges to more effective patient imaging were identified as well as some novel solutions to these challenges.

In the final section of the thesis research, the feasibility of using two of these solutions was investigated with a view to imaging more of the patient's posterior breast volume. Limited angle tomographic trajectories and trajectories that involve raising or lowering the patient bed in mid tomographic acquisition were initially investigated using various geometric phantoms. A very low contrast imaging task was then tested using an observer study to quantify the effect of these trajectories on the ability of observers to maintain visibility of small geometric objects.

This initial integrated SPECT-CT imaging system has demonstrated its ability to successfully perform low dose, dual-modality imaging of the uncompressed breast. Challenges and solutions have been identified here that will make future SPECT-CT designs even more powerful and a clinically relevant technique for molecular imaging of the breast.

Approximately 20-70% of women with breast cancer who choose to undergo breast-conserving surgery (BCS) need to return to the operating table for re-excision [1]. Now devices utilizing optical spectroscopy are emerging as a new platform for intra-operative tumor margin assessment. This study aims to evaluate an optimal decision fusion approach for malignancy detection of measured spectroscopic data from a first-generation optical visible spectral imaging platform that can image the molecular composition of breast tumor margins by implementing a Monte Carlo method with measured diffuse reflectance [1, 2]. The device measures the diffuse reflectance across 450-600nm. After implementing the Monte Carlo algorithm the absorption and scattering spectra is derived and is used to provide insight on different optical properties present in the tissue mass [2]. Although the extracted optical properties may provide insight on the biological composition of a specimen, it may not be ideal for malignancy detection. Demographic factors may also affect a women's normal tissue breast composition, which makes malignancy detection more complicated. This optimal decision fusion approach implements the basic decision fusion methodology on acquired spectroscopic data to evaluate the effect on malignancy detection for different extracted optical parameters. The results of this automated and systematic approach indicate that a performance of 90% sensitivity and 68% specificity can be achieved with this approach for the diffuse reflectance spectrum, which outperforms the extracted optical properties. However, when only considering post-menopausal patients, the absorption spectrum can yield a sensitivity of 90% and specificity of 82% and has the best performance of all other features for this demographic group.

Functional imaging gives clinicians the ability to monitor breast cancer progression and response to therapy. Modern techniques such as Positron Emission Tomography (PET) has allowed for clinicians to visualize the metabolic need of breast cancer and track it longitudinally. However, these techniques are expensive, technologically complex and not easily implemented in rural areas. To add to the difficulty, breast cancer is a highly heterogeneous disease. The heterogeneity means that a single therapy is not always applicable to all patients and every patient requires an individual treatment plan. Being able to first diagnose breast cancer, and then monitor its response to therapy in a cost-effective manner is imperative to improve the survival of patients with this disease.

Optical techniques such as fluorescence are ideal for these applications since they can be fast and implemented with portable technology. These techniques use differences in light interaction with tissue to allow for abnormality detection. This dissertation tests the hypothesis that the fluorescent molecularly specific agents, protoporphyrin IX (PpIX) and 2-NBDG, which utilize metabolic alterations caused by cancer, can be used for ubiquitous breast cancer differentiation and therapy monitoring. Confocal microscopy is used to demonstrate the applicability of both agents in vitro to breast cancer cells regardless of phenotype.

First, 5-aminolevulinic acid (ALA) was incubated with cells causing an increased cellular production of the heme prequel, protoporphyrin IX (PpIX). In cancer cells, the production of PpIX is higher and allows for detection from normal after a 2 hour incubation period. The PpIX was then detected via confocal microscopy and the change in fluorescence intensity between ALA-induced PpIX and controls was measured. A spectroscopy measurement is also completed on a second experimental set of cells to demonstrate that collection of single spectra, post-ALA administration, can discriminate breast cancer cells from normal mammary epithelium.

2-NBDG is a fluorescent glucose analogue that is follows the metabolic pathway of glycolysis, similarly to D-glucose and fluorodeoxyglucose (FDG). Greater accumulation of 2-NBDG can occur in as little as 20 minutes in cells with higher glycolytic demand, which is commonly associated with cancer and hypoxic cells. The shorter incubation period required for 2-NBDG makes it ideal for clinical use, and 2-NBDG was therefore tested further.

2-NBDG uptake was used to detect changes in cellular glycolysis after anti-cancer and endocrine therapy. The anti-cancer therapies, lonidamine and a-cyano-hydroxycinnamate (a-Cinn), which increased and decreased glycolysis, respectively were tested on a subset of breast cancer cells. Lonidamine directly inhibits the metabolism of 2-NBDG and inhibited its uptake. a-Cinn stimulates glycolysis by inhibiting the monocarboxylate transporter 1 preventing lactate from entering as a source for oxidative phosphorylation. 2-NBDG was concurrently increased after a-Cinn treatment. Observation of changes in downstream glycolysis has been determined after the estrogen receptor therapy, tamoxifen, in breast cancer cells. Sixty percent of all breast cancers are estrogen receptor positive (ER+) and have the potential to respond. Known ER+ cells, MCF7, and ER- cells, MDA-MB-435, were treated with tam. 2-NBDG was used to determine therapeutic responders from non-responders by measureable differences in fluorescence uptake.

Finally, the effect of hypoxia, low oxygenation, on 2-NBDG uptake is discussed. The cellular response to hypoxia, known as the Pasteur Effect, causes an increase in glycolysis. Hypoxia is shown in vitro to increase 2-NBDG uptake. Simulated, chronic and cycling hypoxia were completed in vitro with subsequent increases in 2-NBDG as well. Cycling hypoxia has been previously shown to have a greater impact on tumor environment and was implemented in an in vivo murine dorsal window chamber mammary carcinoma model. The uptake of 2-NBDG in tumor and normal tumor-free tissue was tested and 2-NBDG discriminated normal from tumor in a normal oxygen environment. An increase in 2-NBDG was demonstrated after cycling hypoxia in tumor and normal tissue. However, by including hemoglobin saturation data, cycling hypoxic tumor tissue can be discriminated from cycling hypoxic normal tissue and normoxic tumor tissue. From these experiments, the applicability of 2-NBDG as a method to monitor changes in glycolysis and its increased potential by including hemoglobin

saturation measurements is demonstrated.

The progesterone receptor (PR) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. The steroid hormone progesterone binds to PR and induces a conformational change that enables the receptor to bind DNA, recruit cofactors, and directly regulate the transcription of target genes. In addition, extra-nuclear PR can indirectly regulate gene expression by rapidly activating other signaling pathways such as Src/MAPK. Although the direct and indirect functions of PR have been well studied in isolation, it is important to understand the molecular mechanisms by which these pathways can cross talk and integrate to ultimately impact gene expression.

Towards this end, we initiated studies to assess the overall impact of MAPK inhibition on PR transcriptional activity in T47D breast cancer cells treated with the synthetic progestin R5020. During the course of microarray and biochemical analyses that were undertaken to address this issue, we discovered a subset of PR target genes that are enriched for E2F binding sites. Subsequently, we determined that PR-B is a component of several distinct pathways that function both directly and indirectly to positively up-regulate E2F1 expression in T47D breast cancer cells. Firstly, PR directly regulates E2F1 transcription by binding to proximal and distal enhancer sites located near E2F1. Secondly, progestin induces the hyperphosphorylation of Rb, which results in increased recruitment of E2F1 to its own promoter, thereby activating a positive feedback loop that further amplifies its transcription. Finally, PR induces expression of Krüppel-like factor 15 (KLF15) and potentially other Sp/KLF family members, which can bind to GC-rich DNA within the E2F1 promoter and further activate transcription. Together, these results suggest a paradigm for multimodal PR gene regulation that entails cooperation between direct and indirect pathways of PR signaling to achieve the desired downstream transcriptional cascade.

In the breast and other tissues of the female reproductive system, progesterone plays an important role in normal development and function. Therefore, synthetic PR modulators (PRMs) are widely used to manipulate the downstream biology of PR for purposes including contraception and hormone replacement therapy (HRT). However, progestins and PR have also been implicated in disease pathologies such as breast cancer. While the molecular mechanisms by which PR regulates breast tumor growth have not been fully elucidated, recent studies highlight the fact that progestins may have a dose-dependent role in breast cancer progression. Consequently, we undertook studies to identify and characterize any differential effects of low-dose versus high-dose progestins on the downstream activities of PR. Specifically, we found that treatment of breast cancer cells with low-dose progestins can induce maximal transcriptional activation of a subset of PR target genes, including the cell cycle regulators cyclin D1 and E2F1. Furthermore, low-dose and high-dose progestins have differential effects on the phosphorylation of PR and subsequent receptor turnover. Cumulatively, these findings underscore the importance of establishing the effects of a wide range of progestin concentrations on target gene expression and other PR actions, so that we are able to accurately predict the potential consequences of PRMs on downstream PR signaling pathways and biology.

Breast cancer remains among the most common cancers of the developed world. Despite advances in treatment modalities, deaths due to breast cancer are the second leading cause of cancer death among women. The transforming growth factor-beta (TGF-β) pathway is an important modulator of breast cancer progression, acting in a tumor suppressing fashion in early carcinogenesis but switching in a poorly understood fashion to a promoter of cancer progression in later stages. Mutations and loss of function of TGF-β components are common across a variety of cancers. In particular, the expression of the type III TGF-β receptor (TβRIII) is decreased with cancer grade and clinical progression in prostate, lung, ovarian, and pancreatic cancers. In an effort to enhance our understanding of the biology of TGF-β on carcinogenesis, this dissertation looks at the role of TβRIII in breast cancer progression.

Through an examination of clinical specimens, loss of TβRIII was seen at both the message and protein levels with increasing tumor grade. Analysis of correlated patient outcomes showed that low TβRIII expression was predictive of a shorter time to recurrence, demonstrating clinical relevance for TβRIII expression. The contribution of TβRIII to tumor progression was further examined by examining known TGF-β functions, including proliferation, apoptosis, migration, and invasion. TβRIII had no effect on proliferation or apoptosis, but had a suppressive effect on metastasis in vivo, as mammary cancer cells stably expressing TβRIII that were orthotopically injected exhibited lower metatstatic burden and local invasion. In vitro, breast cancer cells exhibited suppression of migration and invasion in transwell assays. Finally, soluble TβRIII (sTβRIII) was shown to recapitulate the suppressive effects on invasion.

To further explore other potential mechanisms by which TβRIII may be mediating its tumor suppressive effects, I examined the contribution of the cytoplasmic domain of TβRIII, which is known to be critical in the regulation of TβRIII cell surface expression and downstream signaling. In vitro, I demonstrated that abrogation of the cytoplasmic domain attenuates the TβRIII-mediated suppression of migration and invasion. TβRIII's suppressive effects are also concomitant with loss of TGF-β signaling, as abrogation of the cytoplasmic domain failed to attenuate TGF-β signaling while the full length receptor was able to do so. In vivo, I also showed that in the absence of the cytoplasmic domain, TβRIII is unable to suppress metastasis and local invasion. Finally, a closer dissection of the cytoplasmic domain revealed that abolishing the interaction of TβRIII with the scaffolding protein GIPC also attenuated TβRIII's ability to dampen TGF-β signaling and invasion.

In sum, TβRIII was established as a prognostic marker for recurrence-free survival of breast cancer patients and as a suppressor of metastasis, migration, and invasion. Furthermore, several mechanisms contribute to TβRIII's tumor suppressive effects, namely the generation of sTβRIII and the interaction of TβRIII with GIPC. Taken together, these studies further demonstrate the importance of TGF-β signaling in cancer biology, elucidate mechanisms by which TβRIII suppresses breast carcinogenesis, and expand upon our understanding of the emerging roles of TβRIII in regulating tumor biology in general.

Apoptotic dysregulation is a hallmark of cancer cells. The inability of cancer cells to undergo apoptosis may lead to therapeutic resistance. Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression and ErbB2 activation. ErbB-targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. Little is known about the molecular mechanisms of therapeutic resistance in IBC cells. We hypothesized that apoptotic dysregulation leads to therapeutic resistance of IBC cells to therapeutic agents, including ErbB-targeting agents. To determine whether apoptotic dysregulation and changes in anti-apoptotic proteins leads to resistance of IBC cells to therapeutic agents, we performed a variety of in vitro-based studies using agents that are used in the clinic to treat IBC patients. The sensitivity of both ErbB2 overexpressing and ErbB1 activated IBC cells to various therapeutic agents was evaluated using various cell death and apoptosis assays, and anti-apoptotic protein expression post-treatment was determined using western blot analysis. The overarching theme observed was that x-linked inhibitor of apoptosis protein (XIAP) expression inversely correlated with sensitivity of cells to therapeutic agents with various mechanisms of action, including TNF-related apoptosis inducing ligand (TRAIL), doxorubicin, cisplatin, paclitaxel, and two ErbB-targeting agents: trastuzumab and a lapatinib-analog (GW583340). Moreover, there was a specific and marked overexpression of XIAP in cells with de novo resistance to trastuzumab and with acquired resistance to GW583340. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. Stable XIAP overexpression using a lentiviral system reversed sensitivity to therapeutic agents (TRAIL and GW583340) in parental IBC cells. Moreover, XIAP downregulation in cells resistant to therapeutic agents (TRAIL, trastuzumab, and GW583340) resulted in decreased viability and increased apoptosis, demonstrating that XIAP is required for survival of cells with resistance to these agents. A novel mechanism of GW583340 oxidative stress-induced mediated apoptosis was identified, and resistant cells had increased antioxidant expression and capability. Interesting, inhibition of XIAP function overcame this increase in antioxidant potential, demonstrating a new function for XIAP in oxidative stress-induced apoptosis. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to therapeutic agents.

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.