Pancreatic cancer is one of the deadliest human malignancies due to its early metastatic spread and resistance to therapy. The mechanisms regulating pancreatic cancer metastasis are so far poorly understood. Here, using both in vitro and in vivo approaches, it is demonstrated that CD44, a transmembrane glycoprotein expressed on a subset of pancreatic cancer cells, is required for the induction of epithelial–mesenchymal transition (EMT) and the activation of an invasive program in pancreatic cancer. Mechanistically, the transcription factor Snail1 (SNAI1), a regulator of the EMT program, is a downstream target of CD44 in primary pancreatic cancer cells and regulates membrane bound metalloproteinase (MMP14/MT1-MMP) expression. In turn, MT1-MMP expression is required for pancreatic cancer invasion. Thus, these data establish the CD44–Snail–MMP axis as a key regulator of the EMT program and of invasion in pancreatic cancer.
Implications: This study sets the stage for CD44 and MT1-MMP as therapeutic targets in pancreatic cancer, for which small molecule or biologic inhibitors are available.
The tumor suppressor p53 (TP53) has a well-studied role in triggering cell-cycle checkpoint in response to DNA damage. Previous studies have suggested that functional p53 enhances chemosensitivity. In contrast, data are presented to show that p53 can be required for cell survival following DNA damage due to activation of reversible cell-cycle checkpoints. The cellular outcome to DNA damage is determined by the duration and extent of the stimulus in a p53-dependent manner. In response to transient or low levels of DNA damage, p53 triggers a reversible G2 arrest, whereas a sustained p53-dependent cell-cycle arrest and senescence follows prolonged or high levels of DNA damage. Regardless of the length of treatment, p53-null cells arrest in G2, but ultimately adapt and proceed into mitosis. Interestingly, they fail to undergo cytokinesis, become multinucleated, and then die from apoptosis. Upon transient treatment with DNA-damaging agents, wild-type p53 cells reversibly arrest and repair the damage, whereas p53-null cells fail to do so and die. These data indicate that p53 can promote cell survival by inducing reversible cell-cycle arrest, thereby allowing for DNA repair. Thus, transient treatments may exploit differences between wild-type p53 and p53-null cells.
Cellular senescence, a form of cell-cycle arrest, is a tumor-suppressor mechanism triggered by multiple tumor-promoting insults, including oncogenic stress and DNA damage. The role of cyclin-dependent kinase 2 (CDK2) regulation has been evaluated in models of replicative senescence, but little is known regarding its role in other senescence settings. Using in vitro and in vivo models of DNA damage–and oncogene-induced cellular senescence, it was determined that activation of the tumor-suppressor protein p53 (TP53) resulted in repression of the CDK2 transcript that was dependent on intact RB. Ectopic CDK2 expression was sufficient to bypass p53-dependent senescence, and CDK2-specific inhibition, either pharmacologically (CVT313) or by use of a dominant-negative CDK2, was sufficient to induce early senescence. Pharmacologic inhibition of CDK2 in an in vivo model of pineal tumor decreased proliferation and promoted early senescence, and it also decreased tumor penetrance and prolonged time to tumor formation in animals lacking p53. In conclusion, for both oncogene- and DNA damage–induced cellular senescence, CDK2 transcript and protein are decreased in a p53- and RB-dependent manner, and this repression is necessary for cell-cycle exit during senescence.
P27Kip1 (CDKN1B) regulates cellular proliferation and senescence, and p27Kip1 deficiency in cancer is strongly correlated with poor prognosis of multiple cancer types. Understanding the mechanism of p27Kip1 loss in cancer and the consequences of restoring p27Kip1 levels is therefore critical for effective management during therapy. Here, SIRT1, a class III histone deacetylase (HDAC), is identified as an important regulator of p27Kip1 expression. Mechanistically, SIRT1 reduces p27Kip1 expression by decreasing p27Kip1 protein stability through the ubiquitin–proteasome pathway. In addition, SIRT1 silencing suppresses non–small cell lung cancer (NSCLC) proliferation and induces senescence in a p27Kip1-dependent manner. Furthermore, SIRT1 silencing dramatically suppresses tumor formation and proliferation in two distinct NSCLC xenograft mouse models. Collectively, these data demonstrate that not only SIRT1 is an important regulator of p27Kip1 but also SIRT inhibition induces senescence and antigrowth potential in lung cancer in vivo.
Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress–dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors.
Despite the frequent expression of N-terminally truncated ErbB2 (NErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how NErbB2 affects chemotherapy-induced cell death. Previously it was shown that NErbB2 upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na+/H+ exchanger (SLC9A1/NHE1) strongly sensitizes NErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which NErbB2 regulates cisplatin-induced breast cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible NErbB2 expression strongly reduced cisplatin-induced ATM- and p53-phosphorylation, augmented Noxa upregulation and caspase-9 and -7 cleavage, doubled p21WAF1/Cip1 (CDKN1A) expression, and nearly abolished Bcl-2 expression. LC3-GFP analysis demonstrated that autophagic flux was reduced by cisplatin in a manner augmented by NErbB2, yet did not contribute to cisplatin-induced death. Using knockdown approaches, it was shown that cisplatin-induced caspase-7 cleavage in NErbB2-MCF-7 cells was Noxa- and caspase-9 dependent. This pathway was augmented by NHE1 inhibition, while the Na+/HCO3– cotransporter (SLC4A7/NBCn1) was internalized following cisplatin exposure.
Ovarian cancer is a lethal disease with the majority of diagnosed women having distant metastases. Interestingly, although Notch3 overexpression has been correlated with poor survival in epithelial ovarian cancer (EOC), little is known about its mechanism of action. Data show that Notch3 specifically promotes anoikis resistance. In addition, data indicate a positive role for focal adhesion kinase (FAK) as well as downstream signaling kinases such as Akt and Erk 1/2 in promoting anchorage-independent growth. Mechanistically, both mRNA transcript and protein levels of type IV collagen (COL4A2) are reduced when Notch3 levels are decreased and exogenous collagen IV supplementation reverses the anoikis sensitivity. Reduction of COL4A2 expression by RNAI-mediated knockdown induces cell death. Finally, elevated Notch3 expression levels correlate with higher COL4A2 expression in human ovarian tumor specimens.
Implications: These data highlight type IV collagen as a novel therapeutic target for metastatic EOC.
Retinoblastoma is the most common pediatric intraocular neoplasm. While retinoblastoma development requires the inactivation of both alleles of the retinoblastoma tumor suppressor gene (RB1) in the developing retina, additional genomic changes are involved in tumor progression, which progressively lead to resistance of tumor cells to death. Therapeutics acting at very downstream levels of death signaling pathways should therefore be interesting in killing retinoblastoma cells. The BH3-only proteins promote apoptosis by modulating the interaction between the pro- and antiapoptotic members of the BCL2 protein family, and this effect can be recapitulated by the BH3 domains. This report analyzes the effect of various BH3 peptides, corresponding to different BH3-only proteins, on two retinoblastoma cell lines, Y79 and WERI-Rb, as well as on the photoreceptor cell line 661W. The BH3 peptide BIRO1, derived from the BCL2L11 death domain, was very effective in promoting Y79 and WERI-Rb cell death without affecting the 661W photoreceptor cells. This cell death was efficient even in absence of BAX and was shown to be caspase independent. While ROS production or AIF release was not detected from mitochondria of treated cells, BIRO1 initiated mitochondria fragmentation in a short period of time following treatment.
Men with metastatic prostate cancer who are treated with androgen deprivation therapies (ADT) usually relapse within 2 to 3 years with disease that is termed castration-resistant prostate cancer (CRPC). To identify the mechanism that drives these advanced tumors, paired-end RNA-sequencing (RNA-seq) was performed on a panel of CRPC bone marrow biopsy specimens. From this genome-wide approach, mutations were found in a series of genes with prostate cancer relevance, including AR, NCOR1, KDM3A, KDM4A, CHD1, SETD5, SETD7, INPP4B, RASGRP3, RASA1, TP53BP1, and CDH1, and a novel SND1:BRAF gene fusion. Among the most highly expressed transcripts were 10 noncoding RNAs (ncRNAs), including MALAT1 and PABPC1, which are involved in RNA processing. Notably, a high percentage of sequence reads mapped to introns, which were determined to be the result of incomplete splicing at canonical splice junctions. Using quantitative PCR (qPCR), a series of genes (AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19) were confirmed to have a greater proportion of unspliced RNA in CRPC specimens than in normal prostate epithelium, untreated primary prostate cancer, and cultured prostate cancer cells. This inefficient coupling of transcription and mRNA splicing suggests an overall increase in transcription or defect in splicing.
The nerve growth factor receptor (NGFR/p75) is a potential tumor suppressor, but its role in colorectal cancer is unknown. Here, the hypermethylation status, biologic function, and clinical relevance were determined for p75NGFR in colorectal cancer. The methylation status and expression of p75NGFR were assessed in colorectal cancer cell lines and clinical tissues by bisulfite genomic sequencing (BGS), qRT-PCR, and immunoblot assay. Methylation of p75NGFR was frequently found in colorectal cancer, leading to its silencing or downregulation, and it was effectively restored by a demethylation agent. The overexpression of p75NGFR in multiple colorectal cancer cell model systems significantly inhibited cell proliferation (concomitant with G1-phase arrest), invasion, and colony formation and induced cell apoptosis. In contrast, p75NGFR knockdown significantly promoted proliferative and invasive phenotypes. Importantly, p75NGFR methylation was observed in the majority of primary colorectal cancer specimens and was associated with histologic grade and preoperative serum CA19-9 levels. Multivariate analysis indicated that patients who lack p75NGFR have reduced overall survival (64% vs. 75%, P = 0.028) and disease-free survival (61% vs. 72%, P = 0.034) compared with p75NGFR-positive patients. In conclusion, p75NGFR is predominantly silenced or downregulated in colorectal cancer, and its biologic activities are consistent with it being a relevant tumor suppressor.
Mechanisms to maintain genomic integrity are essential for cells to remain viable. Not surprisingly, disruption of key DNA damage response pathway factors, such as ataxia telangiectasia-mutated (ATM)/ataxia telangiectasia and RAD3-related (ATR) results in loss of genomic integrity. Here, a synthetic lethal siRNA-screening approach not only confirmed ATM but identified additional replication checkpoint proteins, when ablated, enhanced ATR inhibitor (ATRi) response in a high-content -H2AX assay. Cancers with inactivating ATM mutations exhibit impaired DNA double-stranded break (DSB) repair and rely on compensatory repair pathways for survival. Therefore, impairing ATR activity may selectively sensitize cancer cells to killing. ATR inhibition in an ATM-deficient context results in phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) and leads to induction of -H2AX. Using both in vitro and in vivo models, ATR inhibition enhanced efficacy in ATM loss-of-function mantle cell lymphoma (MCL) compared with ATM wild-type cancer cells. In summary, single-agent ATR inhibitors have therapeutic utility in the treatment of cancers, like MCL, in which ATM function has been lost.
Sarcomatoid metastatic renal cell carcinoma (mRCC) is associated with a poor prognosis, and the biology of the disease has been inadequately characterized. RNA sequencing (RNA-seq) was performed on adjacent benign, clear cell, and sarcomatoid components from clinical specimens with sarcomatoid mRCC. M phase and cell-cycle pathways were enriched in sarcomatoid versus adjacent clear cell components, suggesting greater cell proliferation. The expression of aurora kinase A (AURKA) was increased as part of these pathways, and its increased expression was validated by quantitative PCR (qPCR). Immunohistochemical (IHC) analysis revealed that AURKA levels were increased in sarcomatoid tissue compared with their benign or clear cell parts. The increase in AURKA correlated with increased mTOR pathway activity, as evidenced by increased expression of phosphorylated mTOR (S2448) and ribosomal protein S6K (T389). When AURKA was stably expressed in a RCC cell line (Renca), it resulted in increased expression and activity of mTOR, suggesting that overexpression of AURKA can activate the mTOR pathway. These results warrant the analysis of a larger clinical cohort and suggest that targeting AURKA and/or mTOR in patients with sarcomatoid mRCC should be explored.
Activating mutations of FGFR3 are a common and early event in bladder cancer. Ectopic expression of mutant FGFR3 in normal urothelial cells has both pro-proliferative and antiapoptotic effects at confluence, suggesting that mutant cells are insensitive to cell–cell contact inhibition. Herein, detailed analysis revealed that these cells have reduced cell–cell adhesion, with large intercellular spaces observable at confluence, and diminished cell–substrate adhesion to collagen IV, collagen I, and fibronectin. These phenotypic alterations are accompanied by changes in the expression of genes involved in cell adhesion and extracellular matrix remodeling. Silencing of endogenous mutant FGFR3 in bladder cancer cells induced converse changes in transcript levels of CDH16, PLAU, MMP10, EPCAM, TNC, and HAS3, confirming them as downstream gene targets of mutant FGFR3. Overexpression of EPCAM, HAS3, and MMP10 transcripts was found in a large fraction of primary bladder tumors analyzed, supporting their key role in bladder tumorigenesis in vivo. However, no correlation was found between their protein and/or mRNA expression and FGFR3 mutation status in tumor specimens, indicating that these genes may be targeted by several converging oncogenic pathways. Overall, these results indicate that mutant FGFR3 favors the development and progression of premalignant bladder lesions by altering key genes regulating the cell–cell and cell–matrix adhesive properties of urothelial cells.