Atg7 cooperates with Pten loss to drive prostate cancer tumor growth.

Understanding new therapeutic paradigms for both castrate-sensitive and more aggressive castrate-resistant prostate cancer is essential to improve clinical outcomes. As a critically important cellular process, autophagy promotes stress tolerance by recycling intracellular components to sustain metabolism important for tumor survival. To assess the importance of autophagy in prostate cancer, we generated a new autochthonous genetically engineered mouse model (GEMM) with inducible prostate-specific deficiency in the Pten tumor suppressor and autophagy-related-7 (Atg7) genes. Atg7 deficiency produced an autophagy-deficient phenotype and delayed Pten-deficient prostate tumor progression in both castrate-naïve and castrate-resistant cancers. Atg7-deficient tumors display evidence of endoplasmic reticulum (ER) stress, suggesting that autophagy may promote prostate tumorigenesis through management of protein homeostasis. Taken together, these data support the importance of autophagy for both castrate-naïve and castrate-resistant growth in a newly developed GEMM, suggesting a new paradigm and model to study approaches to inhibit autophagy in combination with known and new therapies for advanced prostate cancer.

Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis.

Macroautophagy (autophagy hereafter) degrades and recycles proteins and organelles to support metabolism and survival in starvation. Oncogenic Ras up-regulates autophagy, and Ras-transformed cell lines require autophagy for mitochondrial function, stress survival, and engrafted tumor growth. Here, the essential autophagy gene autophagy-related-7 (atg7) was deleted concurrently with K-ras(G12D) activation in mouse models for non-small-cell lung cancer (NSCLC). atg7-deficient tumors accumulated dysfunctional mitochondria and prematurely induced p53 and proliferative arrest, which reduced tumor burden that was partly relieved by p53 deletion. atg7 loss altered tumor fate from adenomas and carcinomas to oncocytomas-rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation occurred in atg7-deficient tumors only when p53 was deleted. atg7- and p53-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with p53 loss, Ras-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and autophagy defects may be a molecular basis for the occurrence of oncocytomas. Moreover, cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific.

ERG and CHD1 heterogeneity in prostate cancer: use of confocal microscopy in assessment of microscopic foci.

BACKGROUND: Biomarkers predicting tumor response are important to emerging targeted therapeutics. Complimentary methods to assess and understand genetic changes and heterogeneity within only few cancer cells in tissue will be a valuable addition for assessment of tumors such as prostate cancer that often have insufficient tumor for next generation sequencing in a single biopsy core. METHODS: Using confocal microscopy to identify cell-to-cell relationships in situ, we studied the most common gene rearrangement in prostate cancer (TMPRSS2 and ERG) and the tumor suppressor CHD1 in 56 patients who underwent radical prostatectomy. RESULTS: Wild type ERG was found in 22 of 56 patients; ERG copy number was increased in 10/56, and ERG rearrangements confirmed in 24/56 patients. In 24 patients with ERG rearrangements, the mechanisms of rearrangement were heterogeneous, with deletion in 14/24, a split event in 7/24, and both deletions and split events in the same tumor focus in 3/24 patients. Overall, 14/45 (31.1%) of patients had CHD1 deletion, with the majority of patients with CHD1 deletions (13/14) correlating with ERG-rearrangement negative status (P < 0.001). CONCLUSIONS: These results demonstrate the ability of confocal microscopy and FISH to identify the cell-to-cell differences in common gene fusions such as TMPRSS2-ERG that may arise independently within the same tumor focus. These data support the need to study complimentary approaches to assess genetic changes that may stratify therapy based on predicted sensitivities.

Chronic lymphocytic leukemia modeled in mouse by targeted miR-29 expression.

B-cell chronic lymphocytic leukemia (B-CLL), the most common leukemia in the Western world, occurs in two forms, aggressive (showing for the most part high ZAP-70 expression and unmutated IgH V(H)) and indolent (showing low ZAP-70 expression and mutated IgH V(H)). We found that miR-29a is up-regulated in indolent human B-CLL as compared with aggressive B-CLL and normal CD19(+) B cells. To study the role of miR-29 in B-CLL, we generated Emu-miR-29 transgenic mice overexpressing miR-29 in mouse B cells. Flow cytometric analysis revealed a markedly expanded CD5(+) population in the spleen of these mice starting at 2 mo of age, with 85% (34/40) of miR-29 transgenic mice exhibiting expanded CD5(+) B-cell populations, a characteristic of B-CLL. On average, 50% of B cells in these transgenic mice were CD5 positive. At 2 y of age the mice showed significantly enlarged spleens and an increase in the CD5(+) B-cell population to approximately 100%. Of 20 Emu-miR-29 transgenic mice followed to 24-26 mo of age, 4 (20%) developed frank leukemia and died of the disease. These results suggest that dysregulation of miR-29 can contribute to the pathogenesis of indolent B-CLL.

Effect of dual inhibition of apoptosis and autophagy in prostate cancer.

PURPOSE: Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits autophagy, will increase cytotoxicity of ABT-737. EXPERIMENTAL DESIGN: Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential autophagy gene beclin1. ROS was measured by flow cytometry, and mitophagy assessed by the mCherry-Parkin reporter. RESULTS: Induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cell lines. Therapeutic inhibition of autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity. CONCLUSIONS: We demonstrated that autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of apoptosis and autophagy in future clinical studies.

13q14 deletions in CLL involve cooperating tumor suppressors.

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. 13q14 deletions are most common chromosomal alterations in CLL. We previously reported that miR-15/16 is a target of 13q14 deletions and plays a tumor suppressor role by targeting BCL2. Because DLEU7 is located near miR-15/16 and is also positioned within a minimal deleted region, we investigated whether DLEU7 could also play a tumor suppressor role. Recent studies of transgenic mouse models demonstrated the importance of the nuclear factor-kappaB (NF-kappaB) pathway in CLL. To examine the possible role of DLEU7 in CLL, we investigated the effect of DLEU7 expression on NF-kappaB and nuclear factor of activated T cells (NFAT) activity. We found that DLEU7 functions as a potent NF-kappaB and NFAT inhibitor by physically interacting and inhibiting TACI and BCMA, members of the tumor necrosis factor (TNF) receptor family involved in B-CLL. In addition, DLEU7 expression in A549 lung cancer cells resulted in a decrease in S phase and increased apoptosis. The results suggest that loss of DLEU7 may cooperate with the loss of miR-15/16 in the pathogenesis of CLL.

Tcl1 functions as a transcriptional regulator and is directly involved in the pathogenesis of CLL.

B cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia. Deregulation of the T cell leukemia/lymphoma 1 (TCL1) oncogene in mouse B cells causes a CD5-positive leukemia similar to aggressive human B-CLLs. To examine the mechanisms by which Tcl1 protein exerts oncogenic activity in B cells, we investigated the effect of Tcl1 expression on NF-kappaB and activator protein 1 (AP-1) activity. We found that Tcl1 physically interacts with c-Jun, JunB, and c-Fos and inhibits AP-1 transcriptional activity. Additionally, Tcl1 activates NF-kappaB by physically interacting with p300/CREB binding protein. We then sequenced the TCL1 gene in 600 B-CLL samples and found 2 heterozygous mutations: T38I and R52H. Importantly, both mutants showed gain of function as AP-1 inhibitors. The results indicate that Tcl1 overexpression causes B-CLL by directly enhancing NF-kappaB activity and inhibiting AP-1.

Tal1 transgenic expression reveals absence of B lymphocytes.

TAL1 oncogene encodes a helix-loop-helix transcription factor, Tal1, which is required for blood cell development, and its activation is a frequent event in T-cell acute lymphoblastic leukemia. Tal1 interacts and inhibits other helix-loop-helix factors such as E47 and HEB. To investigate the function of Tal1 in B cells, we generated Emu-TAL1 transgenic mouse line, expressing Tal1 in mouse B-cell lineage. Fluorescence-activated cell sorting (FACS) analysis of lymphocytes isolated from spleens of five out of five founders reveals complete absence of IgM- or CD19-expressing cells. Only 2% to 3% of these cells were B220+ and 100% of B220+ cells were CD43+, indicating that these mice were able to make pro-B cells. Similarly, FACS analysis of bone marrow cells in Emu-TAL1 mice revealed complete absence of B220+IgM+ and B220+CD19+ cells. Analysis of the recombination status of IgH genes revealed the presence of D-J but absence or drastic reduction of V-D-J rearrangements. Our results suggest that Tal1 overexpression in B cells results in a phenotype similar to that of B cells of E47/E2A knockout animals. This represents first in vivo evidence that Tal1 can completely inhibit E47/E2A function.

Tcl1 expression in chronic lymphocytic leukemia is regulated by miR-29 and miR-181.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common human leukemia in the world. Deregulation of the TCL1 oncogene is a causal event in the pathogenesis of the aggressive form of this disease as was verified by using animal models. To study the mechanism of Tcl1 regulation in CLL, we carried out microRNA expression profiling of three types of CLL: indolent CLL, aggressive CLL, and aggressive CLL showing 11q deletion. We identified distinct microRNA signatures corresponding to each group of CLL. We further determined that Tcl1 expression is regulated by miR-29 and miR-181, two microRNAs differentially expressed in CLL. Expression levels of miR-29 and miR-181 generally inversely correlated with Tcl1 expression in the CLL samples we examined. Our results suggest that Tcl1 expression in CLL is, at least in part, regulated by miR-29 and miR-181 and that these microRNAs may be candidates for therapeutic agents in CLLs overexpressing Tcl1.