Sarah J. Freemantle

MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31-mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.

Retinoids in cancer therapy and chemoprevention: promise meets resistance

Retinoids (natural and synthetic derivatives of vitamin A) signal potent differentiation and growth-suppressive effects in diverse normal, premalignant, and malignant cells. A strong rationale exists for the use of retinoids in cancer treatment and chemoprevention based on preclinical, epidemiological, and early clinical findings. Despite the success of all-trans-retinoic acid (RA)-based differentiation therapy in acute promyelocytic leukemia (APL), the broad promise of retinoids in the clinic has not yet been realized. In addition to the expected limited activity of any single therapeutic agent, translation of retinoid activities from the laboratory to the clinic has met with intrinsic or acquired retinoid resistance. Evidence suggests that solid tumors develop intrinsic resistance to retinoids during carcinogenesis. In contrast, relapse of APL is often associated with acquired resistance to retinoid maturation induction. This review discusses what is known about retinoid resistance mechanisms in cancer therapy and chemoprevention. Strategies to overcome this resistance will be discussed, including combination therapy with other differentiation-inducing, cytotoxic or chromatin-remodeling agents, as well as the use of receptor-selective and nonclassical retinoids. Opportunities exist in the post-genomic era to bypass resistance to classical retinoids by identifying target genes and associated pathways that directly mediate the antineoplastic effects of retinoids. In this regard, the retinoids are useful pharmacological tools to reveal important pathways targeted in cancer therapy and chemoprevention.

Retinoic Acid Promotes Ubiquitination and Proteolysis of Cyclin D1 during Induced Tumor Cell Differentiation

Mechanisms by which differentiation programs engage the cell cycle are poorly understood. This study demonstrates that retinoids promote ubiquitination and degradation of cyclin D1 during retinoid-induced differentiation of human embryonal carcinoma cells. In response to all-trans-retinoic acid (RA) treatment, the human embryonal carcinoma cell line NT2/D1 exhibits a progressive decline in cyclin D1 expression beginning when the cells are committed to differentiate, but before onset of terminal neuronal differentiation. The decrease in cyclin D1 protein is tightly associated with the accumulation of hypophosphorylated forms of the retinoblastoma protein and G1 arrest. In contrast, retinoic acid receptor γ-deficient NT2/D1-R1 cells do not growth-arrest or accumulate in G1 and have persistent cyclin D1 overexpression despite RA treatment. Notably, stable transfection of retinoic acid receptor γ restores RA-mediated growth suppression and differentiation to NT2/D1-R1 cells and restores the decline of cyclin D1. The proteasome inhibitor LLnL blocks this RA-mediated decline in cyclin D1. RA treatment markedly accelerates ubiquitination of wild-type cyclin D1, but not a cyclin D1 (T286A) mutant. Transient expression of cyclin D1 (T286A) in NT2/D1 cells blocks RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and represses induction of immunophenotypic neuronal markers. Taken together, these findings strongly implicate RA-mediated degradation of cyclin D1 as a means of coupling induced differentiation and cell cycle control of human embryonal carcinoma cells.

Uncovering Growth-Suppressive MicroRNAs in Lung Cancer

Purpose: MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growth-suppressive miRNAs in lung cancer. Experimental Design: miRNA arrays were done on normal lung tissues and adenocarcinomas from wild-type and proteasome degradation-resistant cyclin E transgenic mice to reveal repressed miRNAs in lung cancer. Real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays validated these findings. Lung cancer cell lines were derived from each transgenic line (designated as ED-1 and ED-2 cells, respectively). Each highlighted miRNA was independently transfected into these cells. Growth-suppressive mechanisms were explored. Expression of a computationally predicted miRNA target was examined. These miRNAs were studied in a paired normal-malignant human lung tissue bank. Results: miR-34c, miR-145, and miR-142-5p were repressed in transgenic lung cancers. Findings were confirmed by real-time and semiquantitative reverse transcription-PCR as well as in situ hybridization assays. Similar miRNA profiles occurred in human normal versus malignant lung tissues. Individual overexpression of miR-34c, miR-145, and miR-142-5p in ED-1 and ED-2 cells markedly repressed cell growth. Anti-miR cotransfections antagonized this inhibition. The miR-34c target, cyclin E, was repressed by miR-34c transfection and provided a mechanism for observed growth suppression. Conclusions: miR-34c, miR-145, and miR-142-5p were repressed in murine and human lung cancers. Transfection of each miRNA significantly repressed lung cancer cell growth. Thus, these miRNAs were growth suppressive and are proposed to exert antineoplastic effects in the lung.

UBE1L is a retinoid target that triggers PML/RARα degradation and apoptosis in acute promyelocytic leukemia

All-trans-retinoic acid (RA) treatment induces remissions in acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, promyelocytic leukemia (PML)/RA receptor α (RARα). Microarray analyses previously revealed induction of UBE1L (ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APL cells. We report here that this occurs within 3 h in RA-sensitive but not RA-resistant APL cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment of the UBE1L promoter was capable of mediating transcriptional response to RA in a retinoid receptor-selective manner. PML/RARα, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of retinoid response in APL is the proteasome-dependent PML/RARα degradation. UBE1L transfection triggered PML/RARα degradation, but transfection of a truncated UBE1L or E1 did not cause this degradation. A tight link was shown between UBE1L induction and PML/RARα degradation. Notably, retroviral expression of UBE1L rapidly induced apoptosis in NB4 APL cells, but not in cells lacking PML/RARα expression. UBE1L has been implicated directly in retinoid effects in APL and may be targeted for repression by PML/RARα. UBE1L is proposed as a direct pharmacological target that overcomes oncogenic effects of PML/RARα by triggering its degradation and signaling apoptosis in APL cells.

Developmentally-related candidate retinoic acid target genes regulated early during neuronal differentiation of human embryonal carcinoma

Embryonal carcinoma is a model of embryonic development as well as tumor cell differentiation. In response to all-trans retinoic acid (RA), the human embryonal carcinoma (EC) cell line, NT2/D1, differentiates toward a neuronal lineage with associated loss of cell growth and tumorigenicity. Through the use of cDNA-based microarrays we sought to identify the early downstream targets of RA during differentiation commitment of NT2/D1 cells. A total of 57 genes were induced and 37 genes repressed by RA. RA regulated genes were restricted at 8 h with 27 genes induced and five repressed. The total number of RA-responsive transcripts increased at 24 and 48 h and their pattern of expression was more symmetrical. For a given time point less than 1% of the 9128 cDNAs on the expression array were regulated by RA. Many of these gene products are associated with developmental pathways including those of TGF-β (Lefty A, NMA, follistatin), homeo domain (HoxD1, Meis2, Meis1, Gbx2), IGF (IGFBP3, IGFBP6, CTGF), Notch (manic fringe, ADAM11), Hedgehog (patched) and Wnt (Frat2, secreted frizzled-related protein 1) signaling. In addition a large cassette of genes induced by RA at 24–48 h are associated with cell adhesion, cytoskeletal and matrix remodeling, growth suppression and intracellular signaling cascades. The majority of repressed genes are associated with protein/RNA processing, turnover or metabolism. The early induced genes identified may play a regulatory role in RA-mediated growth suppression and terminal differentiation and may have physiologic or pharmacologic importance during normal human development and retinoid-based cancer therapy or prevention.

Specific Chemopreventive Agents Trigger Proteasomal Degradation of G 1 Cyclins: Implications for Combination Therapy

Purpose: There is a need to identify cancer chemoprevention mechanisms. We reported previously that all-trans-retinoic acid (RA) prevented carcinogenic transformation of BEAS-2B immortalized human bronchial epithelial cells by causing G1 arrest, permitting repair of genomic DNA damage. G1 arrest was triggered by cyclin D1 proteolysis via ubiquitin-dependent degradation. This study investigated which chemopreventive agents activated this degradation program and whether cyclin E was also degraded. Experimental Design: This study examined whether: (a) cyclin E protein was affected by RA treatment; (b) cyclin degradation occurred in derived BEAS-2B-R1 cells that were partially resistant to RA; and (c) other candidate chemopreventive agents caused cyclin degradation. Results: RA treatment triggered degradation of cyclin E protein, and ALLN, a proteasomal inhibitor, inhibited this degradation. Induction of the retinoic acid receptor β, growth suppression, and cyclin degradation were each inhibited in BEAS-2B-R1 cells. Transfection experiments in BEAS-2B cells indicated that RA treatment repressed expression of wild-type cyclin D1 and cyclin E, but ALLN inhibited this degradation. Mutation of threonine 286 stabilized transfected cyclin D1, and mutations of threonines 62 and 380 stabilized transfected cyclin E, despite RA treatment. Specific chemopreventive agents triggered cyclin degradation. Nonclassical retinoids (fenretinide and retinoid X receptor agonists) and a synthetic triterpenoid (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) each suppressed BEAS-2B growth and activated this degradation program. However, a vitamin D3 analog (RO-24–5531), a cyclooxygenase inhibitor (indomethacin), and a peroxisome proliferator-activated receptor γ agonist (rosiglitazone) each suppressed BEAS-2B growth, but did not cause cyclin degradation. BEAS-2B-R1 cells remained responsive to nonclassical retinoids and to 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid. Conclusions: Specific chemopreventive agents activate cyclin proteolysis. Yet, broad resistance did not occur after acquired resistance to a single agent. This provides a therapeutic rationale for combination chemoprevention with agents activating non-cross-resistant pathways.

Transgenic cyclin E triggers dysplasia and multiple pulmonary adenocarcinomas

Cyclin E is a critical G1-S cell cycle regulator aberrantly expressed in bronchial premalignancy and lung cancer. Cyclin E expression negatively affects lung cancer prognosis. Its role in lung carcinogenesis was explored. Retroviral cyclin E transduction promoted pulmonary epithelial cell growth, and small interfering RNA targeting of cyclin E repressed this growth. Murine transgenic lines were engineered to mimic aberrant cyclin E expression in the lung. Wild-type and proteasome degradation-resistant human cyclin E transgenic lines were independently driven by the human surfactant C (SP-C) promoter. Chromosome instability (CIN), pulmonary dysplasia, sonic hedgehog (Shh) pathway activation, adenocarcinomas, and metastases occurred. Notably, high expression of degradation-resistant cyclin E frequently caused dysplasia and multiple lung adenocarcinomas. Thus, recapitulation of aberrant cyclin E expression as seen in human premalignant and malignant lung lesions reproduces in the mouse frequent features of lung carcinogenesis, including CIN, Shh pathway activation, dysplasia, single or multiple lung cancers, or presence of metastases. This article reports unique mouse lung cancer models that replicate many carcinogenic changes found in patients. These models provide insights into the carcinogenesis process and implicate cyclin E as a therapeutic target in the lung.

Microarray analysis uncovers retinoid targets in human bronchial epithelial cells

Retinoids, the natural and synthetic derivatives of vitamin A, have a role in cancer treatment and prevention. There is a need to reveal mechanisms that account for retinoid response or resistance. This study identified candidate all-trans-retinoic acid (RA) target genes linked to growth suppression in BEAS-2B human bronchial epithelial cells. Microarray analyses were performed using Affymetrix arrays. A total of 11 RA-induced species were validated by reverse transcription polymerase chain reaction (RT-PCR), Western or Northern analyses. Three of these species were novel candidate RA-target genes in human bronchial epithelial cells. These included: placental bone morphogenetic protein (PLAB), polyamine oxidase isoform 1 (PAOh1) and E74-like factor 3 (ELF3). Expression patterns were studied in RA-resistant BEAS-2B-R1 cells. In BEAS-2B-R1 cells, RA dysregulated the expression of the putative lymphocyte G0/G1 switch gene (G0S2), heme oxygenase 1 (HMOX1), tumor necrosis factor-α-induced protein 2 (TNFAIP2), inhibitor of DNA binding 1(Id1), fos-like antigen 1 (FOSL1), transglutaminase 2 (TGM2), asparagine synthetase (ASNS), PLAB, PAOh1 and ELF3, while prominent induction of insulin-like growth-factor-binding protein 6 (IGFBP6) still occurred. In summary, this study identified 11 candidate RA-target genes in human bronchial epithelial cells including three novel species. Expression studies in BEAS-2B-R1 cells indicated that several were directly implicated in RA signaling, since their aberrant expression was linked to RA resistance of human bronchial epithelial cells.

High DNA methyltransferase 3B expression mediates 5-aza-deoxycytidine hypersensitivity in testicular germ cell tumors.

Testicular germ cell tumors (TGCT) are the most common solid tumors of 15- to 35-year-old men. TGCT patients are frequently cured with cytotoxic cisplatin-based therapy. However, TGCT patients refractory to cisplatin-based chemotherapy have a poor prognosis, as do those having a late relapse. Pluripotent embryonal carcinomas (EC) are the malignant counterparts to embryonic stem cells and are considered the stem cells of TGCTs. Here, we show that human EC cells are highly sensitive to 5-aza-deoxycytidine (5-aza-CdR) compared with somatic solid tumor cells. Decreased proliferation and survival with low nanomolar concentrations of 5-aza-CdR is associated with ATM activation, H2AX phosphorylation, increased expression of p21, and the induction of genes known to be methylated in TGCTs (MGMT, RASSF1A, and HOXA9). Notably, 5-aza-CdR hypersensitivity is associated with markedly abundant expression of the pluripotency-associated DNA methyltransferase 3B (DNMT3B) compared with somatic tumor cells. Knockdown of DNMT3B in EC cells results in substantial resistance to 5-aza-CdR, strongly indicating that 5-aza-CdR sensitivity is mechanistically linked to high levels of DNMT3B. Intriguingly, cisplatin-resistant EC cells retain an exquisite sensitivity to low-dose 5-aza-CdR treatment, and pretreatment of 5-aza-CdR resensitizes these cells to cisplatin-mediated toxicity. This resensitization is also partially dependent on high DNMT3B levels. These novel findings indicate that high expression of DNMT3B, a likely byproduct of their pluripotency and germ cell origin, sensitizes TGCT-derived EC cells to low-dose 5-aza-CdR treatment.