EGFR activation is both a key molecular driver of disease progression and the target of a broad class of molecular agents designed to treat advanced cancer. Nevertheless, resistance develops through several mechanisms, including activation of AKT signaling. Though much is known about the specific molecular lesions conferring resistance to anti-EGFR–based therapies, additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. We identified a transcriptional network involving the tumor suppressors Krüppel-like factor 6 (KLF6) and forkhead box O1 (FOXO1) that negatively regulates activated EGFR signaling in both cell culture and in vivo models. Furthermore, the use of the FDA-approved drug trifluoperazine hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restored sensitivity to AKT-driven erlotinib resistance through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft models of lung adenocarcinoma. Combined, these findings define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs as capable of restoring chemosensitivity to anti-EGFR–based therapy for the treatment of metastatic lung adenocarcinoma.
Jaya Sangodkar, Neil S. Dhawan, Heather Melville, Varan J. Singh, Eric Yuan, Huma Rana, Sudeh Izadmehr, Caroline Farrington, Sahar Mazhar, Suzanna Katz, Tara Albano, Pearlann Arnovitz, Rachel Okrent, Michael Ohlmeyer, Matthew Galsky, David Burstein, David Zhang, Katerina Politi, Analisa DiFeo, Goutham Narla
Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.
James L. LaBelle, Samuel G. Katz, Gregory H. Bird, Evripidis Gavathiotis, Michelle L. Stewart, Chelsea Lawrence, Jill K. Fisher, Marina Godes, Kenneth Pitter, Andrew L. Kung, Loren D. Walensky
Telomere shortening limits the proliferative capacity of a cell, but perhaps surprisingly, shortening is also known to be associated with increased rates of tumor initiation. A current hypothesis suggests that telomere dysfunction increases tumor initiation by induction of chromosomal instability, but that initiated tumors need to reactivate telomerase for genome stabilization and tumor progression. This concept has not been tested in vivo, since appropriate mouse models were lacking. Here, we analyzed hepatocarcinogenesis in a mouse model of inducible telomere dysfunction on a telomerase-proficient background, in telomerase knockout mice with chronic telomere dysfunction (G3 mTerc–/–), and in WT mice with functional telomeres and telomerase. Transient or chronic telomere dysfunction enhanced the rates of chromosomal aberrations during hepatocarcinogenesis, but only telomerase-proficient mice exhibited significantly increased rates of macroscopic tumor formation in response to telomere dysfunction. In contrast, telomere dysfunction resulted in pronounced accumulation of DNA damage, cell-cycle arrest, and apoptosis in telomerase-deficient liver tumors. Together, these data provide in vivo evidence that transient telomere dysfunction during early or late stages of tumorigenesis promotes chromosomal instability and carcinogenesis in telomerase-proficient mice.
Yvonne Begus-Nahrmann, Daniel Hartmann, Johann Kraus, Parisa Eshraghi, Annika Scheffold, Melanie Grieb, Volker Rasche, Peter Schirmacher, Han-Wong Lee, Hans A. Kestler, André Lechel, K. Lenhard Rudolph
Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.
Takayuki Hoshii, Yuko Tadokoro, Kazuhito Naka, Takako Ooshio, Teruyuki Muraguchi, Naoyuki Sugiyama, Tomoyoshi Soga, Kimi Araki, Ken-ichi Yamamura, Atsushi Hirao
Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have increased resistance to conventional therapies and are capable of establishing metastasis. However, only a few biomarkers of CSCs have been identified. Here, we report that ganglioside GD2 (a glycosphingolipid) identifies a small fraction of cells in human breast cancer cell lines and patient samples that are capable of forming mammospheres and initiating tumors with as few as 10 GD2+ cells. In addition, the majority of GD2+ cells are also CD44hiCD24lo, the previously established CSC-associated cell surface phenotype. Gene expression analysis revealed that GD3 synthase (GD3S) is highly expressed in GD2+ as well as in CD44hiCD24lo cells and that interference with GD3S expression, either by shRNA or using a pharmacological inhibitor, reduced the CSC population and CSC-associated properties. GD3S knockdown completely abrogated tumor formation in vivo. Also, induction of epithelial-mesenchymal transition (EMT) in transformed human mammary epithelial cells (HMLER cells) dramatically increased GD2 as well as GD3S expression in these cells, suggesting a role of EMT in the origin of GD2+ breast CSCs. In summary, we identified GD2 as a new CSC-specific cell surface marker and GD3S as a potential therapeutic target for CSCs, with the possibility of improving survival and cure rates in patients with breast cancer.
Venkata Lokesh Battula, Yuexi Shi, Kurt W. Evans, Rui-Yu Wang, Erika L. Spaeth, Rodrigo O. Jacamo, Rudy Guerra, Aysegul A. Sahin, Frank C. Marini, Gabriel Hortobagyi, Sendurai A. Mani, Michael Andreeff
Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies resistant to current chemotherapies or radiotherapies, which makes it urgent to identify new therapeutic targets for HCC. In this study, we found that checkpoint kinase 1 (CHK1) was frequently overexpressed and correlated with poor clinical outcome in patients with HCC. We further showed that the CHK1 inhibitor GÖ6976 was capable of sensitizing HCC cells to cisplatin, indicating that CHK1 may have oncogenic function in HCC. We found that CHK1 phosphorylated the tumor suppressor spleen tyrosine kinase (L) (SYK[L]) and identified the phosphorylation site at Ser295. Furthermore, CHK1 phosphorylation of SYK(L) promoted its subsequent proteasomal degradation. Expression of a nonphosphorylated mutant of SYK(L) was more efficient at suppressing proliferation, colony formation, mobility, and tumor growth in HCC lines. Importantly, a strong inverse correlation between the expression levels of CHK1 and SYK(L) was observed in patients with HCC. Collectively, our data demonstrate that SYK(L) is a substrate of CHK1 in tumor cells and suggest that targeting the CHK1/SYK(L) pathway may be a promising strategy for treating HCC.
Jian Hong, Kaishun Hu, Yunfei Yuan, Yi Sang, Qiangui Bu, Guihua Chen, Longjun Yang, Binkui Li, Pinzhu Huang, Dongtai Chen, Yi Liang, Ruhua Zhang, Jingxuan Pan, Yi-Xin Zeng, Tiebang Kang
Vα24-invariant NKT cells inhibit tumor growth by targeting tumor-associated macrophages (TAMs). Tumor progression therefore requires that TAMs evade NKT cell activity through yet-unknown mechanisms. Here we report that a subset of cells in neuroblastoma (NB) cell lines and primary tumors expresses membrane-bound TNF-α (mbTNF-α). These proinflammatory tumor cells induced production of the chemokine CCL20 from TAMs via activation of the NF-κB signaling pathway, an effect that was amplified in hypoxia. Flow cytometry analyses of human primary NB tumors revealed selective accumulation of CCL20 in TAMs. Neutralization of the chemokine inhibited in vitro migration of NKT cells toward tumor-conditioned hypoxic monocytes and localization of NKT cells to NB grafts in mice. We also found that hypoxia impaired NKT cell viability and function. Thus, CCL20-producing TAMs served as a hypoxic trap for tumor-infiltrating NKT cells. IL-15 protected antigen-activated NKT cells from hypoxia, and transgenic expression of IL-15 in adoptively transferred NKT cells dramatically enhanced their antimetastatic activity in mice. Thus, tumor-induced chemokine production in hypoxic TAMs and consequent chemoattraction and inhibition of NKT cells represents a mechanism of immune escape that can be reversed by adoptive immunotherapy with IL-15–transduced NKT cells.
Daofeng Liu, Liping Song, Jie Wei, Amy N. Courtney, Xiuhua Gao, Ekaterina Marinova, Linjie Guo, Andras Heczey, Shahab Asgharzadeh, Eugene Kim, Gianpietro Dotti, Leonid S. Metelitsa
Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all cancers and shows remarkable resistance to cell stress. Nuclear protein 1 (Nupr1), which mediates stress response in the pancreas, is frequently upregulated in pancreatic cancer. Here, we report that Nupr1 plays an essential role in pancreatic tumorigenesis. In a mouse model of pancreatic cancer with constitutively expressed oncogenic KrasG12D, we found that loss of Nupr1 protected from the development of pancreatic intraepithelial neoplasias (PanINs). Further, in cultured pancreatic cells, nutrient deprivation activated Nupr1 expression, which we found to be required for cell survival. We found that Nupr1 protected cells from stress-induced death by inhibiting apoptosis through a pathway dependent on transcription factor RelB and immediate early response 3 (IER3). NUPR1, RELB, and IER3 proteins were coexpressed in mouse PanINs from KrasG12D-expressing pancreas. Moreover, pancreas-specific deletion of Relb in a KrasG12D background resulted in delayed in PanIN development associated with a lack of IER3 expression. Thus, efficient PanIN formation was dependent on the expression of Nupr1 and Relb, with likely involvement of IER3. Finally, in patients with PDAC, expression of NUPR1, RELB, and IER3 was significantly correlated with a poor prognosis. Cumulatively, these results reveal a NUPR1/RELB/IER3 stress-related pathway that is required for oncogenic KrasG12D-dependent transformation of the pancreas.
Tewfik Hamidi, Hana Algül, Carla Eliana Cano, Maria José Sandi, Maria Inés Molejon, Marc Riemann, Ezequiel Luis Calvo, Gwen Lomberk, Jean-Charles Dagorn, Falk Weih, Raul Urrutia, Roland Michael Schmid, Juan Lucio Iovanna
PAX5, a B cell–specific transcription factor, is overexpressed through chromosomal translocations in a subset of B cell lymphomas. Previously, we had shown that activation of immunoreceptor tyrosine-based activation motif (ITAM) proteins and B cell receptor (BCR) signaling by PAX5 contributes to B-lymphomagenesis. However, the effect of PAX5 on other oncogenic transcription factor-controlled pathways is unknown. Using a MYC-induced murine lymphoma model as well as MYC-transformed human B cell lines, we found that PAX5 controls c-MYC protein stability and steady-state levels. This promoter-independent, posttranslational mechanism of c-MYC regulation was independent of ITAM/BCR activity. Instead it was controlled by another PAX5 target, CD19, through the PI3K-AKT-GSK3β axis. Consequently, MYC levels in B cells from CD19-deficient mice were sharply reduced. Conversely, reexpression of CD19 in murine lymphomas with spontaneous silencing of PAX5 boosted MYC levels, expression of its key target genes, cell proliferation in vitro, and overall tumor growth in vivo. In human B-lymphomas, CD19 mRNA levels were found to correlate with those of MYC-activated genes. They also negatively correlated with the overall survival of patients with lymphoma in the same way that MYC levels do. Thus, CD19 is a major BCR-independent regulator of MYC-driven neoplastic growth in B cell neoplasms.
Elaine Y. Chung, James N. Psathas, Duonan Yu, Yimei Li, Mitchell J. Weiss, Andrei Thomas-Tikhonenko
Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis.
Karim Nacerddine, Jean-Bernard Beaudry, Vasudeva Ginjala, Bart Westerman, Francesca Mattiroli, Ji-Ying Song, Henk van der Poel, Olga Balagué Ponz, Colin Pritchard, Paulien Cornelissen-Steijger, John Zevenhoven, Ellen Tanger, Titia K. Sixma, Shridar Ganesan, Maarten van Lohuizen