Blocking Notch signaling in pancreatic cancer promotes hypoxia and cell death.
Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease that is refractory to medical intervention. Notch pathway antagonism has been shown to prevent pancreatic preneoplasia progression in mouse models, but potential benefits in the setting of an established PDA tumor have not been established. We demonstrate that the gamma secretase inhibitor MRK003 effectively inhibits intratumoral Notch signaling in the KPC mouse model of advanced PDA. Although MRK003 monotherapy fails to extend the lifespan of KPC mice, the combination of MRK003 with the chemotherapeutic gemcitabine prolongs survival. Combination treatment kills tumor endothelial cells and synergistically promotes widespread hypoxic necrosis. These results indicate that the paucivascular nature of PDA can be exploited as a therapeutic vulnerability, and the dual targeting of the tumor endothelium and neoplastic cells by gamma secretase inhibition constitutes a rationale for clinical translation.
Glioblastoma (GBM) is the most common malignant brain tumor that is characterized by high proliferative rate and invasiveness. Since dysregualtion of Notch signaling is implicated in the pathogenesis of many human cancers, here we investigated the role of Notch signaling in GBM. We found that there is aberrant activation of Notch signaling in GBM cell lines and human GBM-derived neurospheres. Inhibition of Notch signaling via the expression of a dominant negative form of the Notch co-activator, mastermind-like 1 (DN-MAML1) or the treatment of a γ-secretase inhibitor (GSI) MRK-003 resulted in a significant reduction in GBM cell growth in vitro and in vivo. Knockdown of individual Notch receptors revealed that Notch1 and Notch2 receptors differentially contributed to GBM cell growth, with Notch2 having a predominant role. Furthermore, blockade of Notch signaling inhibited the proliferation of human GBM-derived neurospheres in vitro and in vivo. Our overall data indicate that Notch signaling contributes significantly to optimal GBM growth, strongly supporting that the Notch pathway is a promising therapeutic target for GBM.
Notch signaling; Glioblastoma; Tumor neurospheres; γ-secretase inhibitor; Cell growth
Glioblastoma (GBM) is the most common malignant brain tumor that is characterized by high proliferative rate and invasiveness. Since dysregulation of Notch signaling is implicated in the pathogenesis of many human cancers, here we investigated the role of Notch signaling in GBM. We found that there is aberrant activation of Notch signaling in GBM cell lines and human GBM-derived neurospheres. Inhibition of Notch signaling via the expression of a dominant negative form of the Notch coactivator, mastermind-like 1 (DN-MAML1), or the treatment of a γ-secretase inhibitor, (GSI) MRK-003, resulted in a significant reduction in GBM cell growth in vitro and in vivo. Knockdown of individual Notch receptors revealed that Notch1 and Notch2 receptors differentially contributed to GBM cell growth, with Notch2 having a predominant role. Furthermore, blockade of Notch signaling inhibited the proliferation of human GBM-derived neurospheres in vitro and in vivo. Our overall data indicate that Notch signaling contributes significantly to optimal GBM growth, strongly supporting that the Notch pathway is a promising therapeutic target for GBM.
Notch signaling; glioblastoma; tumor neurospheres; γ-secretase inhibitor; cell growth
Hypoxic microenvironment supports cancer stem cell survival, causes poor response to anticancer therapy and tumor recurrence. Inhibition of Notch-1 signaling in adenocarcinoma of the lung (ACL) cells causes apoptosis specifically under hypoxia. Here we found that Akt-1 activation is a key mediator of Notch-1 pro-survival effects under hypoxia. Notch-1 activates Akt-1 through repression of phosphatase and tensin homolog (PTEN) expression and induction of the Insulin-like Growth Factor 1 Receptor (IGF-1R). The latter seems to be the major determinant of Akt-1 stimulation, since Notch-1 signaling affects Akt-1 activation in PTEN−/− ACL cells. Both downregulation of Insulin Receptor Substrate 1 (IRS-1) and dominant-negative IGF-1R sensitized ACL cells to γ-secretase inhibitor (GSI)-induced apoptosis. Conversely, overexpression of IGF-1R protected ACL cells from GSI toxicity. Inhibition of Notch-1 caused reduced IGF-1R expression, while forced Notch-1 expression yielded opposite effects. ChIP experiments suggested Notch-1 direct regulation of the IGF-1R promoter. Experiments in which human ACL cells were injected in mice confirmed elevated and specific co-expression of Notch-1IC, IGF-1R and pAkt-1 in hypoxic tumor areas.
Our data provide a mechanistic explanation for Notch-1 mediated pro-survival function in hypoxic ACL tumor microenvironment. The results identify additional targets that may synergize with Notch-1 inhibition for ACL treatment.
Notch signaling; lung cancer; hypoxia; IGF-1R; cancer cell survival
The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)—originally developed for Alzheimer's disease—are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)—a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.
The tumor microenvironment (TME) of NSCLC is heterogeneous with variable blood flow though leaky immature vessels, resulting in regions of acidosis and hypoxia. Hypoxia has been documented in NSCLC directly by polarographic needle electrodes and indirectly by assessing tissue and plasma hypoxia markers. In general, elevated expression of these markers portends poorer outcomes in NSCLC. Impaired vascularity and hypoxia can lead to increased metastasis and treatment resistance. Compounds that directly target hypoxic cells such as tirapazamine have been tested in clinical trials for NSCLC with mixed results. Pre-clinical data, however, suggest other ways of exploiting the abnormal TME in NSCLC for therapeutic gain. Inhibition of HIF-1α or VEGF may increase local control after radiation. Inhibitors of the EGFR/PI3K/Akt pathway such as erlotinib or PI-103 may “normalize” tumor vessels, allowing for increased chemotherapy delivery or improved oxygenation and radiation response. In order to select patients who may respond to these therapies and to evaluate the effects of these agents, a non-invasive means of imaging the TME is critical. Presently, there are several promising modalities to image hypoxia and the tumor vasculature; these include dynamic perfusion imaging and positron emission tomography (PET) scanning with radiolabled nitroimidazoles.
TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.
The Notch signaling pathway is a critical embryonic developmental regulatory pathway that has been implicated in oncogenesis. In non-small cell lung cancer (NSCLC), recent evidence suggests that Notch signaling may contribute to maintenance of a cancer stem or progenitor cell compartment required for tumorigenesis. We explored whether intact Notch signaling is required for NSCLC clonogenic and tumorigenic potential in vitro and in vivo using a series of genetically modified model systems. In keeping with previous observations, we find that Notch3 in particular is upregulated in human lung cancer lines, and that down regulation of Notch signaling using a selective γ-secretase inhibitor (MRK-003) is associated with decreased proliferation and clonogenic capacity in vitro. We demonstrate that this phenotype is rescued with the expression of NICD3, a constitutively active cleaved form of Notch3 not affected by γ-secretase inhibition. Using an inducible LSL-KRASG12D model of lung cancer in vivo, we demonstrate a transient upregulation of Notch pathway activity in early tumor precursor lesions. However, a more rigorous test of the requirement for Notch signaling in lung oncogenesis, crossing the LSL-KRASG12D mouse model with a transgenic with a similarly inducible global dominant negative suppressor of Notch activity, LSL-DNMAML (dominant negative mastermind-like), reveals no evidence of Notch pathway requirement for lung tumor initiation or growth in vivo. Distinct Notch family members may have different, and potentially opposing, activities in oncogenesis, and targeted inhibition of individual Notch family members may be a more effective anti-cancer strategy than global pathway suppression.
We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer.
We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo.
Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours.
Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.
ErbB-2; trastuzumab; Notch-1; GSI; recurrence; resistance
Accumulating evidence supports the existence of breast cancer stem cells (BCSCs), which are characterized by their capacity to self-renew and divide indefinitely, and resistance to conventional therapies. The Notch pathway is important for stem cell renewal, and is a potential target for BCSC-directed therapy.
Using human breast tumorgraft studies, we evaluated the impact of gamma secretase inhibitors (GSI) on the BCSC population and the efficacy of combining GSI with docetaxel treatment. The mouse experimental therapy paralleled a concurrent clinical trial in advanced breast cancer patients, designed to determine the maximally tolerated dose of the GSI, MK-0752, administered sequentially with docetaxel, and to evaluate BCSC markers in serial tumor biopsies.
Treatment with GSI reduced BCSCs in MC1 and BMC-2147 tumorgrafts by inhibition of the Notch pathway. GSI enhanced the efficacy of docetaxel in preclinical studies. In the clinical trial, 30 patients with advanced breast cancer were treated with escalating doses of MK-0752 plus docetaxel. Clinically meaningful doses of both drugs were possible, with manageable toxicity and preliminary evidence of efficacy. A decrease in CD44+/CD24−, ALDH+, and MSFE were observed in tumors of patients undergoing serial biopsies.
These preclinical data demonstrate that pharmacological inhibition of the Notch pathway can reduce BCSCs in breast tumorgraft models. The clinical trial demonstrates feasibility of combination GSI and chemotherapy, and together these results encourage further study of Notch pathway inhibitors in combination with chemotherapy in breast cancer.
breast cancer; Phase I clinical trial; cancer stem cells; agents with other mechanisms of action; Notch inhibitors
To examine the effects of clinically relevant pharmacological Notch inhibition on glioblastoma xenografts.
Murine orthotopic xenografts generated from temozolomide sensitive and resistant glioblastoma neurosphere lines were treated with the γ-secretase inhibitor MRK003. Tumor growth was tracked by weekly imaging, and the effects on animal survival and tumor proliferation were assessed, along with the expression of Notch targets, stem cell and differentiation markers, and the biology of neurospheres isolated from previously treated xenografts and controls.
Weekly MRK003 therapy resulted in significant reductions in growth as measured by imaging, as well as prolongation of survival. Microscopic examination confirmed a statistically significant reduction in cross-sectional tumor area and mitotic index in a MRK003-treated cohort as compared to controls. Expression of multiple Notch targets was reduced in the xenografts, along with neural stem/progenitor cell markers, while glial differentiation was induced. Neurospheres derived from MRK003-treated xenografts exhibited reduced clonogenicity and formed less aggressive secondary xenografts. Neurospheres isolated from treated xenografts remained sensitive to MRK003, suggesting that therapeutic resistance does not rapidly arise during in vivo Notch blockade.
Weekly oral delivery of MRK003 results in significant in vivo inhibition of Notch pathway activity, tumor growth, stem cell marker expression and clonogenicity, providing pre-clinical support for the use of such compounds in patients with malignant brain tumors. Some of these effects can persist for some time after in vivo therapy is complete.
Glioblastoma; Cancer Stem Cell; γ-Secretase Inhibitor; Notch
Here, we have investigated the role of the Notch pathway in the generation and maintenance of KrasG12V-driven non-small cell lung carcinomas (NSCLCs). We demonstrate by genetic means that γ-secretase and RBPJ are essential in the formation of NSCLCs. Importantly, pharmacologic treatment of mice carrying autochthonous NSCLCs with a γ-secretase inhibitor (GSI) blocks cancer growth. Treated carcinomas present reduced HES1 levels and, interestingly, reduced phosphorylated ERK without changes in phosphorylated MEK. Mechanistically, we show that HES1 directly binds and represses the promoter of DUSP1, encoding a dual phosphatase active against phospho-ERK. Accordingly, GSI treatment upregulates DUSP1 and decreases phospho-ERK. These data provide proof for the in vivo therapeutic potential of γ-secretase inhibitors in primary NSCLCs.
γ-secretase; NOTCH; KRAS; lung cancer; chemotherapy
Malignant gliomas are treated with a combination of surgery, radiation and temozolomide (TMZ), but these therapies ultimately fail due to tumor recurrence. In glioma cultures, TMZ treatment significantly decreases neurosphere formation; however, a small percentage of cells survive and repopulate the culture. A promising target for glioma therapy is the Notch signaling pathway. Notch activity is upregulated in many gliomas and can be suppressed using gamma-secretase inhibitors (GSIs). Using a neurosphere recovery assay and xenograft experiments, we analyzed if the addition of GSIs with TMZ treatment could inhibit repopulation and tumor recurrence. We demonstrate that TMZ+GSI treatment decreased neurosphere formation and inhibited neurosphere recovery. This enhancement of TMZ treatment occurred through inhibition of the Notch pathway and depended on the sequence of drug administration. In addition, ex vivo TMZ+GSI treatment of glioma xenografts in immunocompromised mice extended tumor latency and survival, and in vivo TMZ+GSI treatment blocked tumor progression in 50% of mice with pre-existing tumors. These data demonstrate the importance of the Notch pathway in chemoprotection and repopulation of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising approach to decrease brain tumor recurrence.
glioma; neurosphere; temozolomide; Notch; gamma-secretase inhibitor
Cholangiocacinoma (CC) is a cancer disease with rising incidence. Notch signaling has been shown to be deregulated in many cancers. However, the role of this signaling pathway in the carcinogenesis of CC is still not fully explored. In this study, we investigated the effects of Notch inhibition by γ-secretase inhibitor IX (GSI IX) in cultured human CC cell lines and we established a transgenic mouse model with liver specific expression of the intracellular domain of Notch (Notch-ICD) and inactivation of tumor suppressor p53. GSI IX treatment effectively impaired cell proliferation, migration, invasion, epithelial to mesenchymal transition and growth of softagar colonies. In vivo overexpression of Notch-ICD together with an inactivation of p53 significantly increased tumor burden and showed CC characteristics. Conclusion: Our study highlights the importance of Notch signaling in the tumorigenesis of CC and demonstrates that additional inactivation of p53 in vivo.
Stromal cells are essential components of the bone marrow (BM) microenvironment that regulate and support the survival of different tumors, including chronic lymphocytic leukemia (CLL). In this study, we investigated the role of Notch signaling in the promotion of survival and chemoresistance of human CLL cells in coculture with human BM-mesenchymal stromal cells (hBM-MSCs) of both autologous and allogeneic origin. The presence of BM-MSCs rescued CLL cells from apoptosis both spontaneously and following induction with various drugs, including Fludarabine, Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone. The treatment with a combination of anti-Notch-1, Notch-2 and Notch-4 antibodies or γ-secretase inhibitor XII (GSI XII) reverted this protective effect by day 3, even in presence of the above-mentioned drugs. Overall, our findings show that stromal cell-mediated Notch-1, Notch-2 and Notch-4 signaling has a role in CLL survival and resistance to chemotherapy. Therefore, its blocking could be an additional tool to overcome drug resistance and improve the therapeutic strategies for CLL.
Notch signaling; human bone marrow mesenchymal stromal cells; hBM-MSCs; chronic lymphocytic leukemia; CLL; drug resistance
Radiotherapy represents the most effective nonsurgical treatments for gliomas. Yet, gliomas are highly radioresistant and recurrence is nearly universal. Results from our laboratory and other groups suggest that cancer stem cells contribute to radioresistance in gliomas and breast cancers. The Notch pathway is critically implicated in stem cell fate determination and cancer. In this study, we showed that inhibition of Notch pathway with gamma-secretase inhibitors (GSIs) rendered the glioma stem cells more sensitive to radiation at clinically relevant doses. GSIs enhanced radiation-induced cell death and impaired clonogenic survival of glioma stem cells, but not non-stem glioma cells. Similarly, knockdown of Notch1 or Notch2 increased radiosensitivity of glioma stem cells. The specificity of the radiosensitizing effects of GSIs was confirmed by expression of the constitutively active intracellular domains of Notch1 or Notch2 that protected glioma stem cells against radiation. Notch inhibition with GSIs did not alter the DNA damage response of glioma stem cells following radiation, but rather impaired radiation-induced Akt activation and upregulated levels of the truncated apoptotic isoform of Mcl-1 (Mcl-1s). Taken together, our results suggest a critical role of Notch to promote radioresistance of glioma stem cells. Inhibition of Notch signaling holds promise to improve the efficiency of current radiotherapy in glioma treatment.
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines, as well as patient-derived PDAC xenografts to determine whether pharmacological targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment is effective against PDAC as evidenced by the down-regulation of nuclear Notch1 intracellular domain (N1ICD), inhibition of anchorage independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pre-treatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared to gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation and induced both apoptosis and intra-tumoral necrosis. Gene expression analysis of untreated tumors indicated that up-regulation of nuclear factor kappa B (NFκB) pathway components were predictive of sensitivity to MRK-003, while up-regulation in B-cell receptor (BCR) signaling and nuclear factor erythroid-derived 2-like 2 (NRF2) pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small molecule inhibition of Notch signaling as a therapeutic strategy in PDAC.
Notch; MRK-003; gamma-secretase inhibitor; pancreatic cancer; chemotherapy
Cancer stem cells (CSCs) play an important role in the development and recurrence of malignant tumors including glioma. Notch signaling, an evolutionarily conserved pathway mediating direct cell-cell interaction, has been shown to regulate neural stem cells (NSCs) and glioma stem cells (GSCs) in normal neurogenesis and pathological carcinogenesis, respectively. However, how Notch signaling regulates the proliferation and differentiation of GSCs has not been well elucidated.
We isolated and cultivate human GSCs from glioma patient specimens. Then on parallel comparison with NSCs, we inhibited Notch signaling using γ-secretase inhibitors (GSI) and assessed the potential functions of Notch signaling in human GSCs.
Similar to the GSI-treated NSCs, the number of the primary and secondary tumor spheres from GSI-treated GSCs decreased significantly, suggesting that the proliferation and self-renewal ability of GSI-treated GSCs were attenuated. GSI-treated GSCs showed increased differentiation into mature neural cell types in differentiation medium, similar to GSI-treated NSCs. Next, we found that GSI-treated tumor spheres were composed of more intermediate progenitors instead of CSCs, compared with the controls. Interestingly, although inhibition of Notch signaling decreased the ratio of proliferating NSCs in long term culture, we found that the ratio of G2+M phase-GSCs were almost undisturbed on GSI treatment within 72 h.
These data indicate that like NSCs, Notch signaling maintains the patient-derived GSCs by promoting their self-renewal and inhibiting their differentiation, and support that Notch signal inhibitor GSI might be a prosperous candidate of the treatment targeting CSCs for gliomas, however, with GSI-resistance at the early stage of GSCs cell cycle.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that the Notch signalling pathway is important in PDAC initiation and maintenance, although the specific cell biological roles of the pathway remain to be established. Here we sought to examine this question in established pancreatic cancer cell lines using the γ-secretase inhibitor IX (GSI IX) to inactivate Notch. Based on the known roles of Notch in development and stem cell biology, we focused on effects on epithelial mesenchymal transition (EMT) and on pancreatic tumor initiating CD44+/EpCAM+ cells. We analyzed the effect of the GSI IX on growth and epithelial plasticity of human pancreatic cancer cell lines, and on the tumorigenicity of pancreatic tumor initiating CD44+/EpCAM+ cells. Notably, apoptosis was induced after GSI IX treatment and EMT markers were selectively targeted. Furthermore, under GSI IX treatment, decline in the growth of pancreatic tumor initiating CD44+/EpCAM+ cells was observed in vitro and in a xenograft mouse model. This study demonstrates a central role of Notch signalling pathway in pancreatic cancer pathogenesis and identifies an effective approach to inhibit selectively EMT and suppress tumorigenesis by eliminating pancreatic tumor initiating CD44+/EpCAM+ cells.
Activating mutations in NOTCH1 are the most prominent genetic abnormality in T-cell acute Lymphoblastic Leukemia (T-ALL) and inhibition of NOTCH1 signaling with γ-secretase inhibitors (GSIs) has been proposed as targeted therapy in this disease. However, most T-ALL cell lines with mutations in NOTCH1 fail to respond to GSI therapy. Using gene expression profiling and mutation analysis we showed that mutational loss of PTEN is a common event in T-ALL and is associated with resistance to NOTCH inhibition. Furthermore, our studies revealed that NOTCH1 induces upregulation of the PI3K-AKT pathway via HES1, which negatively controls the expression of PTEN. This regulatory circuitry is evolutionary conserved from Drosophila to humans as demonstrated by the interaction of overexpression of Delta and Akt in a model of Notch-induced transformation in the fly eye. Loss of PTEN and constitutive activation of AKT in T-ALL induce increased glucose metabolism and bypass the requirement of NOTCH1 signaling to sustain cell growth. Importantly, PTEN-null/GSI resistant T-ALL cells switch their oncogene addiction from NOTCH1 to AKT and are highly sensitive to AKT inhibitors. These results should facilitate the development of molecular therapies targeting NOTCH1 and AKT for the treatment of T-ALL.
T-cell lymphoblastic leukemia; NOTCH1; PTEN; AKT; γ-secretase inhibitor; oncogene addiction
Pancreatic cancer stem cells (CSCs) represent a small subpopulation of pancreatic cancer cells that have the capacity to initiate and propagate tumor formation. However, the mechanisms by which pancreatic CSCs are maintained are not well understood or characterized.
Expression of Notch receptors, ligands, and Notch signaling target genes was quantitated in the CSC and non-CSC populations from 8 primary human pancreatic xenografts. A gamma secretase inhibitor (GSI) that inhibits the Notch pathway and a shRNA targeting the Notch target gene Hes1 were used to assess the role of the Notch pathway in CSC population maintenance and pancreatic tumor growth.
Notch pathway components were found to be upregulated in pancreatic CSCs. Inhibition of the Notch pathway using either a gamma secretase inhibitor or Hes1 shRNA in pancreatic cancer cells reduced the percentage of CSCs and tumorsphere formation. Conversely, activation of the Notch pathway with an exogenous Notch peptide ligand increased the percentage of CSCs as well as tumorsphere formation. In vivo treatment of orthotopic pancreatic tumors in NOD/SCID mice with GSI blocked tumor growth and reduced the CSC population.
The Notch signaling pathway is important in maintaining the pancreatic CSC population and is a potential therapeutic target in pancreatic cancer.
γ-secretase complexes are multisubunit protease complexes that perform the intramembrane cleavage of more than 60 type-I transmembrane proteins, including Notch receptors. Since dysregulated Notch signaling has been implicated in the tumorigenesis and progression of breast cancer, small molecule γ-secretase inhibitors (GSIs) are being tested for their therapeutic potential in breast cancer treatment in several clinical trials. Here, the structure of γ-secretase complex and the development of GSIs are briefly reviewed, the roles of Notch and several other γ-secretase substrates in breast cancer are discussed, and the difference between γ-secretase inhibition and Notch inhibition, as well as the side effects associated with GSIs, are described. A better understanding of molecular mechanisms that affect the responsiveness of breast cancer to GSI might help to develop strategies to enhance the antitumor activity and, at the same time, alleviate the side effects of GSI.
γ-secretase; GSI; Notch; breast cancer
T-cell acute lymphoblastic leukemias and lymphomas (T-ALL) are aggressive hematologic cancers frequently associated with activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of γ-secretase inhibitors (GSIs). Here, we characterized the interaction between PF-03084014, a clinically-relevant GSI, and dexamethasone in preclinical models of glucocorticoid-resistant T-ALL. Combination treatment of the GSI PF-03084014 with glucocorticoids induced a synergistic antileukemic effect in human T-ALL cell lines and primary human T-ALL patient samples. Mechanistically PF-03084014 plus glucocorticoid treatment induced increased transcriptional upregulation of the glucocorticoid receptor and glucocorticoid target genes. Treatment with PF-03084014 and glucocorticoids in combination was highly efficacious in vivo, with enhanced reduction of tumor burden in a xenograft model of T-ALL. Finally, glucocorticoid treatment effectively reversed PF-03084014-induced gastrointestinal toxicity via inhibition of goblet cell metaplasia. These results warrant the analysis of PF-03084014 and glucocorticoids in combination for the treatment of glucocorticoid-resistant T-ALL.
γ-Secretase activity is vital for the transmembrane cleavage of Notch receptors and the subsequent migration of their intracellular domains to the nucleus. Notch overexpression has been associated with breast, colon, cervical and prostate cancers. We tested the effect of three different γ-secretase inhibitors (GSIs) in breast cancer cells. One inhibitor (GSI1) was lethal to breast cancer cell lines at concentrations of 2 μM and above but had a minimal effect on the non-malignant breast lines. GSI1 was also cytotoxic for a wide variety of cancer cell lines in the NCI60 cell screen. GSI1 treatment resulted in a marked decrease in γ-secretase activity and downregulation of the Notch signalling pathway with no effects on expression of the γ-secretase components or ligands. Flow cytometric and western blot analyses indicated that GSI1 induces a G2/M arrest leading to apoptosis, through downregulation of Bcl-2, Bax and Bcl-XL. GSI1 also inhibited proteasome activity. Thus, the γ-secretase inhibitor GSI1 has a complex mode of action to inhibit breast cancer cell survival and may represent a novel therapy in breast cancer.
γ-secretase; breast cancer; Notch; cell cycle arrest; proteasome; apoptosis
Background & Aims
The Notch signaling pathway is required for the expansion of undifferentiated pancreatic progenitor cells during embryonic development and has been implicated in the progression of pancreatic ductal adenocarcinoma (PDAC). The interaction of Notch ligands with their receptors promotes a γ-secretase-dependent cleavage of the Notch receptor and release of the Notch intracellular domain, which translocates to the nucleus and activates transcription. We investigated the role of this pathway in PDAC progression.
We tested the effects of a γ-secretase inhibitor (GSI) that blocks Notch signaling in PDAC cell lines and a genetically engineered mouse model of PDAC (Kras p53 L/+ mice).
Notch signaling was activated in PDAC precursors and advanced tumors. The GSI inhibited the growth of premalignant pancreatic duct-derived cells in a Notchdependent manner. Additionally, in a panel of over 400 human solid tumor-derived cell lines, PDAC cells, as a group, were more sensitive to the GSI than any other tumor type. Finally, the GSI completely inhibited tumor development in the genetically engineered model of invasive PDAC (p<0.005 χ2 test; compared with mice exposed to vehicle).
These results suggest that Notch signaling is required for PDAC progression. Pharmacologic targeting of this pathway offers therapeutic potential in this treatment-refractory malignancy.