Colon cancer is the second leading cause of cancer death. With over 153,000 new CRC cases predicted, it is the third most commonly diagnosed cancer. Early detection can lead to curative surgical intervention, but recurrent and late metastatic disease is frequently treated with chemotherapeutic options based on induction of DNA damage. Understanding mechanism(s) that regulate DNA damage repair within colon tumor cells is essential to developing effective therapeutic strategies. The Notch signaling pathway is known to participate in normal colon development and we have recently described a pathway by which Notch-1, Notch-3 and Smad may regulated EMT and stem-like properties in colon tumor cells, promoting tumorigenesis. Little is known about how Notch may regulate drug resistance. In this study, we used shRNA to generate colon tumor cells with loss of Notch-3 expression. These cells exhibited reduced expression of the base-excision repair proteins PARP-1 and APE1, along with increased sensitivity to ara-c and cisplatin. These data point to a pathway in which Notch-3 signaling can regulate DNA repair within colon tumor cells and suggests that targeting Notch-3 may be an effective approach to rendering colon tumors sensitive to chemotherapeutic drugs.

Colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways such as Notch and TGF-β contribute to these phenomena. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-β signaling pathway. We hypothesized that Smad3, mediates the Notch-induced stemness and EMT observed in CRC cells. The human colorectal carcinoma cell line HCT-116, stably transduced with constitutively active Notch-1 (ICN) or a GFP-vector control was treated with different combinations of TGF-β1, DAPT (a Notch inhibitor), or SIS3 (a Smad3 inhibitor). Western blot analysis was performed to determine the effects of Smad3 stimulation and inhibition on Notch and potential downstream EMT-related targets, CD44, Slug and Snail. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression and effectively inhibited CD44, Slug, and Snail expression. Colosphere forming ability was also reduced in cells with inhibited Smad3. These results indicate a key role of TGF-β signaling in Notch1-induced tumorigenesis, and suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.

The GSK-3 kinases, GSK-3α and GSK-3β, have a central role in regulating multiple cellular processes such as glycogen synthesis, insulin signaling, cell proliferation and apoptosis. GSK-3β is the most well studied, and was originally described for its role in regulating glycogen synthase. GSK-3β has been studied as a participant in the oncogenic process in a variety of cancers due to its intersection with the PTEN/PI3K/AKT and RAS/RAF/MEK/ERK pathways. Dysregulated signaling through the Notch family of receptors can also promote oncogenesis. Normal Notch receptor signaling regulates cell fate determination in stem cell pools. GSK-3β and Notch share similar targets such β-catenin and the WNT pathway. WNT and β-catenin are involved in several oncogenic processes including those of the colon. In addition, GSK-3β may directly regulate aspects of Notch signaling. This review describes how crosstalk between GSK-3β and Notch can promote oncogenesis, using colon cancer as the primary example.

Many undergraduate students in the health professions experience group work throughout their educational development, but few have the opportunity to experience teamwork in the professional disciplines. Interprofessional teamwork is necessary for successful delivery of accessible, high-quality healthcare aimed at achieving good clinical outcomes and operational efficiencies. Developing teamwork skills early in health professions education results in more successful teams in the workplace. Implementing project-based curricula that integrate undergraduate research and teamwork can lead to discipline-specific learning. The Biomedical Sciences (BMD) and Health Care Management (HCM) undergraduate majors in the UAB School of Health Professions (SHP) each focus on a distinct arm of the healthcare system. To provide these students with interprofessional training early in their education as emerging health professionals, the SHP honors curricula were revised to provide research-based experiential learning and team-building opportunities. Our approach engages BMD and HCM students in interprofessional teams in partnership with faculty mentors and community health providers to complete discipline-specific research projects that ultimately solve real-world problems, resulting in development of critical thinking, team skills, leadership, and cultural awareness of healthcare.

Abstract A role for cancer initiating cells (CIC) in tumor growth, drug resistance, relapse, and metastasis via epithelial to mesenchymal transition (EMT) has been supported by experimental evidence. It is well-documented that Notch-1 receptor signaling participates in stem cell maintenance and lineage commitment in various organs, including the colon, and can regulate EMT. The function of Notch signaling in colorectal cancer (CRC) is not clear. We have reported that Notch-1 activation in the human colon tumor cell line HCT-116 resulted in upregulation of Jagged-1, Smad-3, CD44, and Slug proteins. These changes were accompanied by acquisition of a mesenchymal cell phenotype that included increased anchorage independent growth and migratory activity. Experiments with a γ-secretase inhibitor (DAPT) and soluble Jagged1-Fc protein demonstrated that Notch-1 signaling activates CD44 and Slug via other Notch receptors. In subsequent experiments, we observed an increase in Notch-3 expression in the presence of activated Notch-1. Herein, we tested the hypothesis that the stem cell-like mesenchymal phenotype induced by Notch-1 signaling in colon cancer cells is mediated by the Notch-3 receptor and Smad-3. The human colon tumor cell line HCT-116 was transduced with constitutively active Notch-1, and a Notch-3 shRNA to produce a Notch-3 null cell line with activated Notch-1 (ICN1-shN3 cells). ICN1-shN3 cells exhibited a 2.5-fold lower plating efficiency than the HCT-116/ICN1 cell line (p<0.01) that expresses constitutively active Notch-1 in the presence of Notch-3. In addition, Notch-3 null cells with constitutively active Notch-1 (ICN1-shN3 cells) were slower by 29% in completing wound healing as compared with the HCT-116/ICN1 cell line (p<0.05). Consistent with the lower plating efficiency, a 37% decrease in colosphere formation was observed with ICN1-shN3 cells as compared with the HCT-116/ICN1 cell line (p<0.001). These data indicated that loss of Notch-3 abrogated the Notch-1 induced mesenchymal cell phenotype. Western blot analysis demonstrated that the HCT-116/ICN1 cells with intact Notch-3 exhibited increased levels of the EMT associated proteins Smad-3, CD44 and Slug as compared to the parental HCT-116 cell line. Expression of CD44 and Slug in Notch-3 receptor null ICN1-shN3 cells was highly reduced compared with HCT-116/ICN1 cells and was similar to that detected in the parental HCT-116 cells. Treatment of HCT-116/ICN1 cells with a Smad-3 inhibitor resulted in significant reduction of the CD44 and Slug proteins. Meanwhile, Notch-3 receptor null ICN1-shN3 cells were also more sensitive to chemotherapy treatment than the HCT-116/ICN1 cell line (p<0.05). Collectively, our data provide evidence for a novel pathway in which Notch-1 signaling in colon tumor cells promotes a mesenchymal phenotype via activation of Jagged-1, Notch-3 and Smad-3, followed by expression of CD44 and Slug. Citation Format: George Sigounas, Fred E. Bertrand, Douglas A. Weidner, Kaitlyn E. Vinson, Alexander G. Clark, Azeem Khan. Notch-1 promotes a mesenchymal phenotype in colon cancer cells via Notch-3 and Smad-3 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 856. doi:10.1158/1538-7445.AM2017-856

Colorectal cancer (CRC) is the third leading cause of cancer death worldwide. It is also the third most common cancer diagnosis among men, and the second most common cancer diagnosis among women. Globally, CRC can account for nearly 694,000 annual deaths. It is widely appreciated that CRC is the result of dysregulated cellular pathways that promote an inappropriate stem‐cell‐like phenotype, apoptotic resistance, unchecked proliferation and metastatic spread. While no single pathway is responsible for all of these attributes, an array of recent studies suggests a pivotal role for abnormal Notch‐1 signaling in CRC, in part due to interconnectivity of Notch with other pathways. This review will summarize recent evidence for a role of Notch signaling in CRC, will consider interconnectivity between Notch and other pathways involved in CRC and will discuss the possible utility of targeting Notch as a CRC therapeutic.

Colorectal cancer (CRC) is the third leading cause of cancer death in the United States, resulting in an average of 50,000 deaths per year. Surgery and combination chemotherapy comprise current treatment strategies. However, curative options are limited if surgery and chemotherapy are unsuccessful. Several studies have indicated that CRC aggressiveness and potential for metastatic spread are associated with the acquisition of stem cell like properties. The Notch-1 receptor and its cognate signaling pathway is well known for controlling cell fate decisions and stem-cell phenotypes. Alterations in Notch receptors and Notch signaling has been reported for some colon cancers. Herein, we examine a potential role for Notch-1 signaling in CRC. In CRC patient samples, Notch-1 expression was increased in colon tumor tissue as compared with normal colon tissue. Retroviral transduction of constitutively active Notch-1 (ICN1) into the colon tumor cell line HCT-116 resulted in increased expression of the EMT/stemness associated proteins CD44, Slug, Smad-3, and induction of Jagged-1 expression. These changes in ICN1 expressing cells were accompanied by increased migration and increased anchorage independent growth by 2.5-fold and 23%, respectively. Experiments with the pan-Notch inhibitor DAPT, and soluble Jagged-1-Fc protein provided evidence that Notch-1 signaling activates CD44, Slug, and Smad-3 via a cascade of other Notch-receptors through induction of Jagged-1 expression. These data indicate a key role for Notch signaling in the phenotype of CRC and suggest that targeting of Notch signaling may be of therapeutic value in colon cancers.

Over the past decade, our understanding of the role that Notch-signaling has in tumorigenesis has shifted from leukemogenesis into cancers of solid tumors. Emerging data suggests that in addition to direct effects mediated through the canonical Notch pathway, Notch may participate in epithelial tumor development through regulation of pathways such as PTEN/PI3K/Akt. Prostate cancer is a disease for which PTEN gene expression is especially essential. This review will summarize a role for Notch in prostate development and cancer with an emphasis on how the Notch pathway may intersect with PTEN/PI3K/Akt and mTOR signaling.

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified and studied in the regulation of glycogen synthesis. GSK-3 functions in a wide range of cellular processes. Aberrant activity of GSK-3 has been implicated in many human pathologies including: bipolar depression, Alzheimer's disease, Parkinson's disease, cancer, non-insulin-dependent diabetes mellitus (NIDDM) and others. In some cases, suppression of GSK-3 activity by phosphorylation by Akt and other kinases has been associated with cancer progression. In these cases, GSK-3 has tumor suppressor functions. In other cases, GSK-3 has been associated with tumor progression by stabilizing components of the beta-catenin complex. In these situations, GSK-3 has oncogenic properties. While many inhibitors to GSK-3 have been developed, their use remains controversial because of the ambiguous role of GSK-3 in cancer development. In this review, we will focus on the diverse roles that GSK-3 plays in various human cancers, in particular in solid tumors. Recently, GSK-3 has also been implicated in the generation of cancer stem cells in various cell types. We will also discuss how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTORC1, Ras/Raf/MEK/ERK, Wnt/beta-catenin, Hedgehog, Notch and others.

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death nationwide. In 2013, nearly 156,000 patients will be diagnosed with CRC. Surgical resection for early stage disease offers the best chance for cure; however, up to 30% of patients will relapse. Current models of CRC progression hold that tumor development is driven by a small subpopulation of cancer initiating (CIC) or cancer stem (CSC) cells that share many properties of stem cells. These cells have the capacity to drive tumor growth, are largely responsible for relapse and have been implicated in metastasis via epithelial to mesenchymal transition (EMT), similar to normal counterparts in organogenesis. Notch-1 is the major receptor for a highly conserved signaling pathway that is well-documented to control stem cell and lineage commitment in various organs, including the colon. The function of Notch signaling in colorectal cancer, however, is not well-defined. Based on preliminary data in which colon tumor samples from CRC patients expressed Notch-1 but surrounding benign tissue did not, we sought to test the hypothesis that Notch-1 signaling in colon cells promotes tumorigenesis by enhancing stem cell and EMT characteristics of tumor cells. The human colon tumor line HCT-116 was transduced with a retroviral construct that expresses constitutively active Notch-1 (ICN1). Hes-1 protein levels were increased in the activated Notch-1 transduced cells, indicating that the construct was functional in activating Notch-signaling. These HCT116/ICN1 cells exhibited higher plating efficiency than the parental cell line. The average colony size of the HCT116/ICN1 cells was about two-fold smaller than the parental cell line (p<0.001). In addition, cells with constitutively active Notch-1 (HCT116/ICN1) had a longer duplication time than parental HCT-116 cells (p<0.03). Consistent with longer duplication time, HCT116/ICN1 cells were two-fold slower in completing wound healing as compared with the parental HCT-116 cell line (p<0.001). Transwell migration assays revealed a two-fold increase in migration of HCT116/ICN1 cells as compared with the parental control (p<0.03). Western blot analysis demonstrated that the HCT116/ICN1 cells with activated Notch-1 expressed highly increased levels of the EMT associated proteins Smad3, CD44 and Slug. This was accompanied by a four-fold decreased expression of E-cadherin. Meanwhile, E-cadherin was not altered in HCT116 cells transduced with a Notch-1 shRNA lentiviral construct that inactivated Notch-1 signaling. Finally, levels of the numb protein, a Notch-1 antagonist, appeared to be significantly reduced (five-fold decrease) by constitutive Notch-1 signaling. Alternatively, inactivation of Notch-1 resulted in a four-fold increased expression of the numb protein. Collectively, our data point to the Notch-1 pathway as being a potential key regulator of EMT in colorectal tumors and as such, may be of therapeutic or prognostic utility. Citation Format: Alex Fender, Makenzie Nutter, Timothy Fitzgerald, Fred Bertrand, George Sigounas. Notch-1 regulated stemness and EMT in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1952. doi:10.1158/1538-7445.AM2014-1952

Prostate cancer affects one in three men over the age of 60. Loss of expression or function of PTEN is the most commonly observed molecular defect in human prostate cancer. Thirty to 70% of clinical cases exhibit loss of this critical tumor suppressor. PTEN is an essential negative regulator of PI3K/Akt signaling. Together with the mTOR pathway, the PTEN/PI3K/Akt cascade forms a network for cellular responses to growth factors and nutrients. Studies in mice have demonstrated that small changes in PTEN dose can influence cancer development. Thus, understanding the regulation of PTEN expression and function is key to understanding tumor progression. We have previously reported that signaling through the Notch-1 receptor pathway results in increased transcriptional expression of the tumor suppressor PTEN. We further reported that Notch-1 signaling was lost in the tumor foci of clinical prostate cancer cases as compared to the surrounding benign tissue. Herein, we report that Notch-1 signaling alters the pool of active, unphosphorylated PTEN in the human metastatic prostate tumor cell line DU145. Retroviral transduction was used to generate DU145 cells that express constitutively active Notch-1 (DU/ICN1). In cells with constitutively active Notch-1 (DU/ICN1), the pool of unphosphorylated PTEN was increased roughly 2.5 fold compared to the vector only control. Constitutive Notch-1 signaling also resulted in decreased mTOR signaling as determined by a decrease in phosphorlyated 4E-BP1 and phosphorylated S6 ribosomal protein. Decreased expression of Raptor and Rictor and decreased phosphorylation of mTOR were also observed in the presence of constitutively active Notch-1. To test if Notch-1 signaling influences tumor engraftment and growth in a syngeneic model, the tumorigenic C2 cell line, derived from TRAMP mice, was transduced with constitutively active Notch-1. C2/ICN1 and parental C2 cells were engrafted into C57/BL6 mice. All recipients of the parental C2 cell line developed tumors within 46 days, whereas none that received cells expressing constitutively active Notch-1 (C2/ICN1) had developed tumors. Only after 61 days did one of the four mice that received C2/ICN1 cells develop tumors. We next tested if loss of Notch-1 expression promotes characteristics associated with tumorigenicity by using lentiviral transduction to knock down endogenous Notch-1 expression in DU145 cells (DU/shN1). DU145 cells with loss of Notch-1 (DU/shN1) exhibited decreased expression of PTEN protein and a decreased ability to migrate in transwell experiments as compared with control cells expressing endogenous Notch-1. Collectively, these data indicate a role for Notch-1 receptor signaling in modulating the activity of PI3K/Akt and mTOR axis through regulation of the PTEN tumor suppressor, and suggest a mechanistic basis for Notch-1 tumor suppressive activity in prostate cells. Citation Format: Jennifer M. Nutter, C William Angus, Fred E. Bertrand. Notch-1 regulation of the PTEN - mTOR axis in prostate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1566. doi:10.1158/1538-7445.AM2014-1566

A hallmark of cancer is reactivation/alteration of pathways that control cellular differentiation during developmental processes. Evidence indicates that WNT, Notch, BMP and Hedgehog pathways have a role in normal epithelial cell differentiation, and that alterations in these pathways accompany establishment of the tumorigenic state. Interestingly, there is recent evidence that these pathways are intertwined at the molecular level, and these nodes of intersection may provide opportunities for effective targeted therapies. This review will highlight the role of the WNT, Notch, BMP and Hedgehog pathways in colon cancer.

A hallmark of cancer is reactivation/alteration of pathways that control cellular differentiation during developmental processes. Evidence indicates that WNT, Notch, BMP and Hedgehog pathways have a role in normal epithelial cell differentiation, and that alterations in these pathways accompany establishment of the tumorigenic state. Interestingly, there is recent evidence that these pathways are intertwined at the molecular level, and these nodes of intersection may provide opportunities for effective targeted therapies. This review will highlight the role of the WNT, Notch, BMP and Hedgehog pathways in colon cancer.

Abstract Notch-1 is part of an evolutionarily ancient cell surface receptor signaling pathway that functions in the determination of stem/progenitor cell fate. The human genome encodes four Notch-receptors (Notch-1, -2, -3, and -4) and at least five Notch-ligands (Delta-1, -3, -4, Jagged-1, and-2). Signaling is initiated upon ligand binding to the Notch receptor, which results in a series of proteolytic cleavages that release constitutively active intracytoplamsic Notch (ICN). Nuclear translocation of ICN results in activation of the CBF-1 transcription factor which in turn increases expression of developmentally regulated transcription factors such as Hes-1 and Hey-1. The PTEN tumor suppressor gene is a recently identified Notch-1 target. We have reported that Notch-1 signaling increases expression of the PTEN tumor suppresser gene in prostate tumor cell lines and that Notch-1 signaling is lost in the tumor foci of prostate adenocarcinoma patients. To test the hypothesis that loss of Notch-1 will result in deregulation of normal prostate epithelial cell differentiation and promote tumorigenesis, a lentiviral vector bearing a Notch-1 shRNA (shN1) was used to stably knockdown Notch-1 expression in the benign human prostate epithelial cell line, RWPE-1. Western blotting revealed that RWPE-1/shN1 cells lacked Notch-1 expression, and exhibited reduced expression of Hes-1, confirming that loss of Notch-1 expression resulted in loss of Notch signaling. PTEN protein expression was also reduced, consistent with our previous studies demonstrating that Notch-1 signaling regulated PTEN expression. RT-PCR experiments verified that loss of PTEN expression was mediated at the level of transcription. RWPE-1/shN1 cells lacking Notch-1 expression exhibited a more rounded morphology, as compared with the parental cell line. Rhodamine-phalloidin staining revealed that RWPE-1/shN1 cells had less numerous and shorter cell surface actin projections. RWPE-1/shN1 cells lacking Notch-1 expression exhibited a 2-fold increase in acinar differentiation upon 3-D culture in matrigel as compared with the parental cell line. Flow cytometric analysis revealed that RWPE-1 cells without Notch-1 expression had reduced surface expression of TROP2 and CD149f. Preliminary RT-PCR results indicate that expression of the basal cell marker keratin-5 is reduced in RWPE-1/shN1 cells as compared with parental RWPE-1 cells. Our data suggest that loss Notch-1 expression, and hence loss of signaling, may promote developmental plasticity in prostate epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 497. doi:10.1158/1538-7445.AM2011-497

Automated classification of biological cells according to their 3D morphology is highly desired in a flow cytometer setting. We have investigated this possibility experimentally and numerically using a diffraction imaging approach. A fast image analysis software based on the gray level co-occurrence matrix (GLCM) algorithm has been developed to extract feature parameters from measured diffraction images. The results of GLCM analysis and subsequent classification demonstrate the potential for rapid classification among six types of cultured cells. Combined with numerical results we show that the method of diffraction imaging flow cytometry has the capacity as a platform for high-throughput and label-free classification of biological cells.

Abstract Gamma-glutamyl transferase (GGT1) is a cell membrane bound enzyme that functions in gamma-glutamyl cycling, to provide cells with intracellular glutathione (GSH). GGT1 has been recognized to participate in chemotherapeutic resistance of tumors to carboplatin and various alkylating agents by increasing GSH levels intracellularly, thus allowing the tumor to escape electrophilic damage. Cysteinylglycine generated by membrane bound GGT1 can bind to extracellular cisplatin and prevent it from entering the cell. Mechanisms that target GGT1 and alter its function have been postulated to increase tumor sensitivity to conventional chemotherapies. We have generated a novel monoclonal antibody (mAb), 8G9 [IgG2a, k1] that recognizes GGT1. An immunohistochemical (IHC) survey of benign adult human tissues revealed an antigen distribution in formalin-fixed, paraffin embedded tissues consistent with previous reports of GGT1 expression. A subsequent IHC screen of nearly 100 kidney tumors revealed that scoring based on 8G9 immunoreactivity showed a preference for clear cell (94.45%), multilocular cystic (100%) and papillary cell (95.83%) kidney tumors. A statistical association of 8G9 immunostaining with Fuhrman nuclear grade or pathology staging was not evident. FACS analysis confirmed that 8G9 recognizes a cell surface epitope. Cross-immunoprecipitations performed with 8G9 and a commercially available GGT1 antibody confirmed that 8G9 recognizes GGT1. 8G9 exhibited a unique preference for native GGT1 likely through recognition of a combined epitope formed by the association of the GGT1 heavy and light chains. Because 8G9 appears to recognize a native, surface accessible GGT1 epitope, we performed xenograft studies to test the efficacy of 8G9 in blocking kidney tumor growth either as a single agent or in combination with cisplatin or carboplatin. Athymic nude mice were engrafted with the human kidney tumor cell line Caki-1. Upon successful tumor engraftment, mice were treated with 8G9, cisplatin or carboplatin as single agents or with 8G9+ ciplatin or 8G9 + carboplatin. As a single agent, 8G9 treatment did not result in a reduction of relative tumor volume. Likewise, there was little difference in relative tumor volume in mice treated a combination of 8G9 and carboplatin. However, 8G9 + cisplatin treatment exhibited a modest reduction in tumor volume. These preliminary data suggest that targeting GGT1 may have a cooperative effect in reducing tumor growth in the presence of cisplatin, and that the efficacy of chemotherapy combined with targeting GGT1 may be particularly sensitive to the mechanistic action of the chemotherapeutic agent. Further studies are needed to fully evaluate GGT1 as a potential drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3640. doi:10.1158/1538-7445.AM2011-3640

FDTD modeling and angle-resolved measurement of Mueller matrix elements have been conducted with suspensions of two white blood cell lines at three wavelengths. We found that S12exhibits the largest difference.