ALDH+ tumor-initiating cells exhibiting gain in NOTCH1 gene copy number have enhanced regrowth sensitivity to a γ-secretase inhibitor and irinotecan in colorectal cancer

The Notch signaling pathway has been shown to be upregulated in colorectal cancer (CRC) and important for the self-renewal of cancer stem cells. In this study, we evaluated the efficacy of PF-03084014, a γ-secretase inhibitor, in combination with irinotecan to identify the effects of treatment on tumor recurrence and the tumor-initiating population in our CRC preclinical explant model. The combination of PF-03084014 and irinotecan had the greatest effect at reducing tumor growth on four CRC tumors when compared with treatment with PF-03084014 or irinotecan alone. The combination significantly reduced tumor recurrence in two CRC explants (CRC001 and CRC036) after treatment was discontinued. Both of these tumors exhibited elevated baseline levels of Notch pathway activation as well as an increase in NOTCH1 gene copy number when compared with the two CRC explants (CRC026 and CRC027) where tumors reappeared quickly after termination of treatment. Isolation and injection of aldehyde dehydrogenase (ALDH(+) and ALDH(-)) cells in an in vivo explant model demonstrated that the ALDH(+) cell population were tumorigenic. Evaluation of the ALDH(+) cells after 28 days of treatment showed that the combination reduced the ALDH(+) population in the tumors that did not regrow. Furthermore, ALDH(+) cells from CRC001 and CRC027 were injected in vivo and treated immediately for 28 days. Two months after treatment, tumors were evident in the combination treatment group for CRC027 but not for CRC036. These results indicate the combination of PF-03084014 and irinotecan may be effective in reducing tumor recurrence in CRC patients whose tumors exhibit elevated levels of the Notch pathway.     

Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a γ-secretase inhibitor,in a preclinical colorectal explant model

Background:

Dysregulation of the Notch pathway has been identified to play an important role in the development and progression of colorectal cancer (CRC). In this study, we used a patient-derived CRC explant model to investigate the efficacy of the clinical γ-secretase inhibitor (GSI) PF-03084014.

Methods:

A total of 16 CRC explants were treated with PF-03084014. Knockdown of RBPjκ gene was used to determine the specificity of PF-03084014. Evaluation of the Notch and Wnt pathways in CRC explant tumours was performed by gene array and immunoblotting.

Results:

We identified a subset of CRC tumours that exhibited elevations of the Notch and Wnt pathways sensitive to PF-03084014. Treatment with the GSI resulted in a significant reduction in cleaved Notch, Axin2 (Wnt-dependent gene) and active β-catenin. In addition, knockdown of the RBPjκ gene showed that PF-03084014 has specificity for the Notch pathway in an HCT116 cell line xenograft model. Finally, an increase in apoptosis was observed in CRC001- and CRC021-sensitive tumours.

Conclusion:

This study provides evidence that inhibition of γ-secretase may be beneficial in a subset of patients with elevated levels of the Wnt and Notch pathways.     

Sensitization of Pancreatic Cancer Stem Cells to Gemcitabine by Chk1 Inhibition

Checkpoint kinase 1 (Chk1) inhibition sensitizes pancreatic cancer cells and tumors to gemcitabine. We hypothesized that Chk1 inhibition would sensitize pancreatic cancer stem cells to gemcitabine. We tested this hypothesis by using two patient-derived xenograft models (designated J and F) and the pancreatic cancer stem cell markers CD24, CD44, and ESA. We determined the percentage of marker-positive cells and their tumor-initiating capacity (by limiting dilution assays) after treatment with gemcitabine and the Chk1 inhibitor, AZD7762. We found that marker-positive cells were significantly reduced by the combination of gemcitabine and AZD7762. In addition, secondary tumor initiation was significantly delayed in response to primary tumor treatment with gemcitabine + AZD7762 compared with control, gemcitabine, or AZD7762 alone. Furthermore, for the same number of stem cells implanted from gemcitabine- versus gemcitabine + AZD7762-treated primary tumors, secondary tumor initiation at 10 weeks was 83% versus 43%, respectively. We also found that pS345 Chk1, which is a measure of DNA damage, was induced in marker-positive cells but not in the marker-negative cells. These data demonstrate that Chk1 inhibition in combination with gemcitabine reduces both the percentage and the tumor-initiating capacity of pancreatic cancer stem cells. Furthermore, the finding that the Chk1-mediated DNA damage response was greater in stem cells than in non-stem cells suggests that Chk1 inhibition may selectively sensitize pancreatic cancer stem cells to gemcitabine, thus making Chk1 a potential therapeutic target for improving pancreatic cancer therapy.     

Targeting IL‐13Rα2 in human pancreatic ductal adenocarcinoma with combination therapy of IL‐13‐PE and gemcitabine

Pancreatic cancer is an aggressive disease with only limited therapeutic options available. We have identified that 71% pancreatic ductal adenocarcinoma (PDA) express high levels of IL‐13Rα2, a high‐affinity receptor for IL‐13. To target IL‐13Rα2, we have developed a recombinant immunotoxin, which is a fusion of IL‐13 and Pseudomonas exotoxin (IL‐13‐PE). Since IL‐13‐PE and a commonly used cytotoxic drug gemcitabine act by a different mechanism, we hypothesized that they synergize in mediating antitumor response. Both IL‐13‐PE and gemcitabine‐mediated cytotoxicity to two pancreatic cancer cell lines and when combined synergistic cytotoxicity was observed. This synergism was also demonstrated in vivo in an orthotopic mouse model of human PDA. IL‐13‐PE and gemcitabine showed complete eradiation of tumors as assessed by whole body imaging of GFP‐transfected tumors in 57% of mice in an early cancer model resulting into prolongation of survival. In contrast, monotherapy with either agent did not produce complete eradiation, but tumor volumes were significantly decreased. In advanced PDA model, combination therapy also produced dramatic reduction in tumor growth and enhanced survival compared to animals treated with either agent alone. When IL‐13Rα2 was knocked‐down by RNAi prior to tumor implantation, IL‐13‐PE and gemcitabine did not synergize indicating that IL‐13Rα2 is essential. Mechanistically, gemcitabine increased IL‐13Rα2 expression in vitro and in vivo, which resulted in a synergism of combination therapy. Interestingly, PDA cancer stem cells were resistant to gemcitabine, but not to IL‐13‐PE. These results suggest that combination therapy with IL‐13‐PE and gemcitabine may be a useful strategy for PDA therapy.     

Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells

Despite evidence supporting an oncogenic role in breast cancer, the Notch pathway's contribution to metastasis remains unknown. Here, we report that the Notch ligand Jagged1 is a clinically and functionally important mediator of bone metastasis by activating the Notch pathway in bone cells. Jagged1 promotes tumor growth by stimulating IL-6 release from osteoblasts and directly activates osteoclast differentiation. Furthermore, Jagged1 is a potent downstream mediator of the bone metastasis cytokine TGFβ that is released during bone destruction. Importantly, γ-secretase inhibitor treatment reduces Jagged1-mediated bone metastasis by disrupting the Notch pathway in stromal bone cells. These findings elucidate a stroma-dependent mechanism for Notch signaling in breast cancer and provide rationale for using γ-secretase inhibitors for the treatment of bone metastasis.     

Interleukin-4 cytotoxin therapy synergizes with gemcitabine in a mouse model of pancreatic ductal adenocarcinoma

Targeting cell surface receptors with cytotoxins or immunotoxins provides a unique opportunity for tumor therapy. Here, we show the efficacy of the combination therapy of gemcitabine with an interleukin-4 (IL-4) cytotoxin composed of IL-4 and truncated Pseudomonas exotoxin in animal models of pancreatic ductal adenocarcinoma (PDA). We have observed that 42 of 70 (60%) tumor samples from patients with PDA express moderate- to high-density surface IL-4 receptor (IL-4R), whereas normal pancreatic samples express no or low-density IL-4R. IL-4 cytotoxin was specifically and highly cytotoxic [50% protein synthesis inhibition (IC50) ranging from >0.1 to 13 ng/mL] to six of eight pancreatic cancer cell lines, whereas no cytotoxicity (IC50>1,000 ng/mL) was observed in normal human pancreatic duct epithelium cells, fibroblasts, and human umbilical vein endothelial cells (HUVEC). We also showed that IL-4 cytotoxin in combination with gemcitabine exhibited synergistic antitumor activity in vitro. To confirm synergistic antitumor activity in vivo and monitor precise real-time disease progression, we used a novel metastatic and orthotopic mouse model using green fluorescent protein-transfected cancer cells and whole-body imaging system. The combination of both agents caused complete eradication of tumors in 40% of nude mice with small established PDA tumors. In addition, combined treatment significantly prolonged the survival of nude mice bearing day 14 advanced distant metastatic PDA tumors. Similar results were observed in mice xenografted with PDA obtained from a patient undergoing surgical resection. These results indicate that IL-4 cytotoxin combined with gemcitabine may provide effective therapy for the treatment of patients with PDA.     

Delta like ligand 4 induces impaired chemo-drug delivery and enhanced chemoresistance in pancreatic cancer

The stubborn chemoresistance of pancreatic ductal adenocarcinoma (PDA) is simultaneously influenced by tumor parenchymal and stromal factors, and the ctritical role of Notch ligand Delta-like 4 (DLL4) in the regulation of tumor malignancies has been observed. DLL4 positive expression ratio between duct cells from clinical tumor and adjacent tissues was statistically significant, and the overactivation of DLL4/Notch pathway enhanced the phenotype of EMT and cancer stem cell, even can induce multi-chemoresistance in vitro. Notably, the accompanied defective angiogenesis directly induced inefficient chemo-drug delivery in vivo. Collectively, overexpressed DLL4 on neoplastic cells can enhance chemoresistance through angiogenesis-dependent/independent mechanisms in PDA.     

The FAK scaffold inhibitor C4 disrupts FAK-VEGFR-3 signaling and inhibits pancreatic cancer growth

Even with successful surgical resection and perioperative chemotherapy and radiation, pancreatic ductal adenocarcinoma (PDA) has a high incidence of recurrence. Tumor cell survival depends on activation of signaling pathways that suppress the apoptotic stimuli of invasion and metastasis. Focal adhesion kinase (FAK) is a critical signaling molecule that has been implicated in tumor cell survival, invasion and metastasis. We have previously shown that FAK and vascular endothelial growth factor receptor 3 (VEGFR-3) are overexpressed in cancer cells and physically interact to confer a significant survival advantage. We subsequently identified a novel small molecule inhibitor C4 that targeted the VEGFR-3-FAK site of interaction. In this study, we have shown that C4 disrupted the FAK-VEGFR-3 complexes in PDA cells. C4 treatment caused dose-dependent dephosphorylation and inactivation of the VEGFR-3 and FAK, reduction in cell viability and proliferation, cell cycle arrest and apoptosis in PDA cells. C4 increased the sensitivity of tumor cells to gemcitabine chemotherapy in vitro that lead to apoptosis at nanomolar concentrations of both drugs. C4 reduced tumor growth in vivoin subcutaneous and orthotopic murine models of PDA. The drug alone at low dose, decreased tumor growth; however, concomitant administration with low dose of gemcitabine had significant synergistic effect and led to 70% tumor reduction. Combination of C4 with gemcitabine had a prolonged cytostatic effect on tumor growth after treatment withdrawal. Finally, we report an anecdotal case of stage IV pancreatic cancer treated with gemcitabine in combination with C4 that showed a significant clinical response in primary tumor and complete clinical response in liver metastasis over an eight month period. Taken together, these results demonstrate that targeting the scaffolding function of FAK with a small-molecule FAK-VEGFR-3 inhibitor can be an effective therapeutic strategy against PDA.     

nab-Paclitaxel potentiates gemcitabine activity by reducing cytidine deaminase levels in a mouse model of pancreatic cancer

Nanoparticle albumin-bound (nab)-paclitaxel, an albumin-stabilized paclitaxel formulation, demonstrates clinical activity when administered in combination with gemcitabine in patients with metastatic pancreatic ductal adenocarcinoma (PDA). The limited availability of patient tissue and exquisite sensitivity of xenografts to chemotherapeutics have limited our ability to address the mechanistic basis of this treatment regimen. Here, we used a mouse model of PDA to show that the coadministration of nab-paclitaxel and gemcitabine uniquely demonstrates evidence of tumor regression. Combination treatment increases intratumoral gemcitabine levels attributable to a marked decrease in the primary gemcitabine metabolizing enzyme, cytidine deaminase. Correspondingly, paclitaxel reduced the levels of cytidine deaminase protein in cultured cells through reactive oxygen species-mediated degradation, resulting in the increased stabilization of gemcitabine. Our findings support the concept that suboptimal intratumoral concentrations of gemcitabine represent a crucial mechanism of therapeutic resistance in PDA and highlight the advantages of genetically engineered mouse models in preclinical therapeutic trials. SIGNIFICANCE: This study provides mechanistic insight into the clinical cooperation observed between gemcitabine and nab-paclitaxel in the treatment of pancreatic cancer.     

Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas

Pancreatic ductal adenocarcinoma (PDA) remains a devastating disease with nearly equal incidence and mortality rates. Over the past few decades, a litany of randomized clinical trials has failed to improve the outcome of this disease. More recently, the combination chemotherapy regimen FOLFIRINOX has shown improvement in overall survival over the single agent gemcitabine, and nab-paclitaxel (an albumin-coated formulation of paclitaxel) in combination with gemcitabine has shown promising results in phase II studies. Despite limited impact on patient care as of yet, the molecular and biologic understanding of PDA has advanced substantially. This includes understanding the genomic complexity of the disease, the potential importance of the tumor microenvironment, the metabolic adaptation of PDA cells to obtain nutrients in a hypoxic environment, and the role of pancreatic cancer stem cells. These fundamental discoveries are starting to be translated into clinical studies. In this overview, we discuss the implications of biologic understanding of PDA in clinical research and provide insights for future development of novel approaches and agents in this disease.     

Notch信号通路与消化系统肿瘤相关性的研究进展

Notch信号是一种遗传进化上高度保守,反映相邻细胞间通信作用的一种信号通路,其不仅在细胞正常发育、分化增殖和凋亡中起着重要的作用,而且与多种肿瘤的发生和发展具有相关性.食管鳞状细胞癌的发生常伴随Notch-1的低表达,但食管腺癌的发生却与Notch信号的高表达相关,且高表达的Notch信号对胃癌形成具有促进作用,其表达量提高的程度预示胃癌形成风险的高低.结肠癌中Notch-1表达的升高与病理分级、淋巴转移和病程相关,而Nctch配体Dll-4可促进结肠癌中新生血管的生成有助于癌细胞的转移和远端浸润,与之相反的是Notch-2却可能起到抑制结肠癌生长的作用.总之,目前Notch信号多被视为致癌因素,可促进肿瘤的生长,但在某些肿瘤中也能够诱导肿瘤细胞分化、抑制肿瘤细胞的增殖,表现为致癌与抑癌两种截然相反的作用.应用γ-分泌酶抑制剂(γ-secretase inhibitor,GSI)、小RNA干扰技术和单克隆抗体等方法阻断Notch信号通路,将成为肿瘤治疗的一个新方向.     

Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.     

MUC4 down-regulation reverses chemoresistance of pancreatic cancer stem/progenitor cells and their progenies

The present study was undertaken to estimate the therapeutic benefit to down-regulate the MUC4 mucin for reversing chemoresistance of pancreatic cancer (PC) stem/progenitor cells and their progenies. The results have revealed that MUC4 mucin is overexpressed in CD133⁺ and CD133⁻ pancreatic cells (PCs) detected in patient’s adenocarcinoma tissues while no significant expression was seen in normal pancreatic tissues. The gain- and loss-of-function analyses have indicated that the overexpression of MUC4 in PC lines is associated with a higher resistance to the anti-proliferative, anti-invasive and apoptotic effects induced by gemcitabine. Importantly, the treatment of the MUC4-overexpressing CD18/HPAF-Src cells with gemcitabine resulted in an enrichment of the side population (SP) cells expressing CD133 while the total PC cells including non-SP cells detected in MUC4 knockdown CD18/HPAF-shMUC4 cells were responsive to the cytotoxic effects induced by gemcitabine. These data suggest that the MUC4 down-regulation may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate the total PC cell mass, and thereby preventing disease relapse.     

Cyclopamine increases the cytotoxic effects of paclitaxel and radiation but not cisplatin and gemcitabine in Hedgehog expressing pancreatic cancer cells

Introduction: The hedgehog signaling pathway (Hh) is frequently over expressed in pancreatic adenocarcinomas. We studied the potential cytotoxic interactions between cyclopamine, a Hh pathway inhibitor and paclitaxel, cisplatin, gemcitabine and ionizing radiation (IR). Methods: In vitro clonogenic survival analysis was performed with cyclopamine alone or cyclopamine in combination with paclitaxel, gemcitabine, cisplatin and IR in Hh expressing human pancreatic tumor cells and Hh non-expressing colon cancer cells. Relative cytotoxicity was assessed in combination treatment compared with exposure to single agents. Assays of apoptosis (annexin V) were performed in the presence of cyclopamine, chemotherapeutic agents, and IR. Results: We report that cyclopamine increased the cytotoxic effects of paclitaxel and IR in Hh expressing pancreatic carcinoma cells. These effects were not observed in Hh non-expressing cells. Cyclopamine did not significantly increase killing by cisplatin or gemcitabine in Hh expressing pancreatic cancer cells. Conclusions: These data suggest strategies to combine Hh inhibitors with radiotherapy and chemotherapeutic agents, specifically paclitaxel and related compounds in the treatment of pancreatic cancer.Z. Shafaee and H. Schmidt contributed equally to this work.     

TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner

Our neoadjuvant clinical trial of a GM-CSF secreting allogeneic pancreas tumor vaccine (GVAX) revealed the development of tertiary lymphoid aggregates (TLAs) within the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment 2 weeks after GVAX treatment. Microarray studies revealed that multiple components of the TGF-β pathway were suppressed in TLAs from patients who survived greater than 3 years and who demonstrated vaccine-enhanced mesothelin-specific T cell responses. We tested the hypothesis that combining GVAX with TGF-β inhibitors will improve the anti-tumor immune response of vaccine therapy. In a metastatic murine model of pancreatic cancer, combination therapy with GVAX vaccine and a TGF-β blocking antibody improved the cure rate of PDA-bearing mice. TGF-β blockade in combination with GVAX significantly increased the infiltration of effector CD8⁺ T lymphocytes, specifically anti-tumor-specific IFN-γ producing CD8⁺ T cells, when compared to monotherapy controls (all p < 0.05). TGF-β blockade alone did not deplete T regulatory cells (Tregs), but when give in combination with GVAX, GVAX induced intratumoral Tregs were depleted. Therefore, our PDA preclinical model demonstrates a survival advantage in mice treated with an anti-TGF-β antibody combined with GVAX therapy and provides strong rational for testing this combinational therapy in clinical trials.     

Notch versus the proteasome: what is the target of γ-secretase inhibitor-I?

γ-Secretase inhibitors are new anti-cancer agents targeting Notch signaling. Their specificity for Notch is as yet unclear. Han and colleagues investigated the effects of Z-LeuLeuNleu-CHO on growth of breast cancer cells. The results demonstrated a reduction in cell viability primarily via proteasome inhibition independent of Notch activity. Currently, γ-secretase inhibitors in clinical trials are structurally distinct from Z-LeuLeuNleu-CHO. Their effects on the proteasome are yet to be determined. However, findings from Han and colleagues pose two critical questions: Is the level of proteasomal activity in breast tumors the driving force for growth? What does the Notch pathway contribute to this growth?Breast cancer continues to be the second leading cause of cancer-related deaths among women. In a recent article in Breast Cancer Research, Han and colleagues investigated the effects of γ-secretase inhibitor (GSI)-I on growth of genetically different breast cancer cells [1].Along with GSIs, current therapeutic strategies are increasingly promising - particularly those targeted at ErbB-2 (HER2⁺)-positive or estrogen receptor alpha (ERα⁺)-positive breast tumors. Triple-negative (HER2-negative, ERα-negative, and progesterone receptor-negative) or basal-like breast cancers lack targeted treatment, demonstrating the highest mortality rate and overall reduced disease-free survival [2]. Emerging treatment approaches to help increase overall patient survival lie in targeting novel pathways for the specific subtypes of breast cancer. New chemotherapeutics aim at targeting the Notch signaling pathway, one of the developmental cell-to-cell contact transmembrane proteins involved in cell fate determination, in cell differentiation, in cell proliferation, and possibly in tumor-initiating cells [3]. Notch-1 and its ligand, Jagged-1, have been shown to be co-overexpressed in breast cancer and to be correlated with the poorest overall survival [4], and thus the signaling pathway has surfaced as a potential therapeutic target.The Notch pathway has a critical cleavage step involving a complex of four proteins called the γ-secretase complex. Enzymatic cleavage of Notch by the γ-secretase complex, the third proteolytic cleavage in the pathway, is essential for the formation of the active intracellular Notch domain: this is therefore a desirable step for targeted inhibition. GSIs are still in their infancy, leaving desired mechanistic effects necessary to elucidate.In the previous issue of Breast Cancer Research, Han and colleagues investigated the targeted effects of GSI-I [1]. Their findings using GSI-I, a drug aimed at inhibiting the Notch pathway, demonstrated cell death independent of Notch - uncovering the proteasome as its main target. The effects of GSIs recently developed and/or used in clinical trials are currently being studied to understand their action on breast cancer cell death. Similarly, proteasome inhibitors are a recent line of anticancer drugs that have been shown to cause cancer cell death and are still currently being investigated [5].Using several breast cancer cell lines, Han and colleagues demonstrated that common GSIs such as DAPT and L-685,458 inhibited γ-secretase and intracellular Notch domain formation, but had no effect on cell viability and death. With Z-LeuLeuNleu-CHO (GSI-I), however, they were able to induce cell death via proteasomal inhibition and reduce γ-secretase activity. Interestingly, they observed no apparent effects of DAPT or L-685,458 on the proteasome. The authors therefore concluded that Z-LeuLeuNleu-CHO promoted cell death predominantly through proteasome inhibition. In contrast, a study by Farnie and colleagues found that DAPT reduced mammosphere formation by 22% compared with vehicle [6]. In accordance, Meurette and colleagues demonstrated that DAPT reduced Akt phosphorylation in MCF-7 and DCIS cell lines and increased sensitivity to the chemotherapeutic class of nitrogen mustard alkylating agents, known as melphalan (Alkeran) [7]. These studies indicate that DAPT inhibited tumor-initiating breast cancer cell growth and increased sensitivity to chemotherapeutic agents. The importance of inhibiting the Notch pathway might therefore be context dependent.The authors do comment on the discrepancies of their findings compared with previous published literature on the basis of employing different experimental strategies. GSI-I induced G₂/M arrest and apoptosis in breast cancer cell lines with effects on both γ-secretase activity and the proteasome [8]. In agreement with Han and colleagues'' results, similar studies indicated that GSI-I inhibits HER2-overexpressing breast tumor-initiating sphere formation, which was not apparent in MCF-7 cells that express low to moderate levels of HER2 [9]. Interestingly, Han and colleagues showed that proteasome inhibition using lactacystin reduced cell viability in ERα^-SKBr3 cells and MDA-MB-231 cells, with little effect on ERα⁺MCF-7 cells. Could this observation suggest that the level of proteasome activity differs between breast cancer subtypes and that the degree to which GSI-I functions as a proteasome inhibitor would depend on high proteasome activity? With this in mind, their novel findings unfurled a potential mechanism that could be advantageous.Han and colleagues'' results could suggest a potential duality in therapeutic treatment of breast cancer cells. Proteasome inhibition could be a vital target of treatment in combination with other oncogenic or growth-promoting proteins. Evidence suggests the importance of inhibiting the Notch pathway in combination with current targeting of ERα [10] or HER2 [11]. For example, recent studies have shown that GSI-I or Ly 411,575 treatment of triple-negative MDA-MB-231 cells or treatment in combination with tamoxifen in ERα⁺T47D:A18 cells arrested growth and caused tumor regression in vivo, respectively [10]. In the HER2⁺ breast cancer cell line, BT474, Ly 411,575 or MRK-003 GSI treatment increased apoptosis and re-sensitized resistant HER2⁺ cells to trastuzumab [12]. Furthermore, these two studies showed that specific knockdown of Notch-1 by siRNA inhibited cell proliferation and increased sensitivity to either 4-hydroxy-tamoxifen or trastuzumab. These articles demonstrated the importance of Notch-1 signaling in ERα^-, ERα⁺, or HER2⁺ breast cancer cells [10,12]. GSIs are therefore currently in clinical trials as anticancer drugs, but specific pharmacologic and molecular properties are still being investigated.Because little is known about which GSIs will be the most advantageous in the clinic, it is important to understand structural and biochemical differences between specific compounds. Han and colleagues used the Z-LeuLeuNleu-CHO GSI, which is a derivative of the proteasome inhibitor MG-132. The structure of MG-132 is Z-LeuLeuLeu-CHO. GSI-I is very close structurally, differing only by a single norleucine substitution: Z-LeuLeuNorLeu-CHO. Treatment with two structurally similar drugs targeted at different enzymes - that is, γ-secretase or the proteasome - could therefore be anticipated to inherently have shared effects, which could account for the results on the proteasome seen by GSI-I.Furthermore, current GSIs that are being used in clinical trials are a new line of medicinally and structurally modified chemical molecules that could show different effects. DAPT and L-685,458 GSIs, which were used in Han and colleagues'' article, are not used in current trials and are structurally unique. Structurally modified GSIs are still being investigated and could provide evidence for cellular death via inhibition of the Notch pathway independent of the proteasome. Understanding these key properties may lead to better therapeutic targeting. Studying the effect of these new classes of GSIs in clinical trials on proteasome activity could prove very interesting.In conclusion, in the present article we comment that Z-LeuLeuLeu-CHO is a derivative of a proteasomal inhibitor, which could inherently have shared effects; that current GSIs used in clinical trials are structurally unique and their effects on the proteasome are as yet unknown; and that the level of proteasomal activity, which could differ between breast cancer subtypes, may play a critical role by which GSI-I functions to inhibit growth. In summary, Han and colleagues beg the intriguing question of whether proteasome inhibitors independently or in combination with GSIs or with current chemotherapeutic agents provide novel and exciting therapeutic strategies against breast cancer.     

Synergistic effects of acyclic retinoid and gemcitabine on growth inhibition in pancreatic cancer cells

Pancreatic cancer is a serious healthcare problem worldwide because of its high mortality. Gemcitabine, a DNA synthesis inhibitor, is the standard first-line treatment for advanced pancreatic cancer and is also expected as a key drug for the combination therapy of this malignancy. Retinoids, which are derivatives of vitamin A, exert anti-tumor effects in various types of human malignancies, including pancreatic cancer. This study examined whether combination therapy with gemcitabine and acyclic retinoid (ACR), a new synthetic retinoid, had enhanced anti-tumor efficacy in pancreatic cancer. ACR, 9-cis-retinoic acid and gemcitabine preferentially inhibited the growth of human pancreatic cancer cells (Panc-1 and KP-2) in comparison to PE normal human pancreatic epithelial cells. The combination of ACR plus gemcitabine synergistically inhibited the growth of Panc-1 cells. The combined treatment with these two agents also acted synergistically to induce apoptosis and to inhibit Ras activation in these cancer cells. In vivo, the combination therapy augmented tumor growth inhibition through the induction of apoptosis and inhibition of cell proliferation in tumor tissue. These results suggest that the combination of ACR plus gemcitabine may therefore be an effective regimen for the chemotherapy of pancreatic cancer.