Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18 to 25 nucleotides) with probable roles in the regulation of gene expression. In Caenorhabditis elegans, lin-4 and let-7 miRNAs control the timing of fate specification of neuronal and hypodermal cells during larval development. lin-4, let-7 and other miRNA genes are conserved in mammals, and their potential functions in mammalian development are under active study. AbstractBackground: The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18 to 25 nucleotides) with probable roles in the regulation of gene expression. In Caenorhabditis elegans, lin-4 and let-7 miRNAs control the timing of fate specification of neuronal and hypodermal cells during larval development. lin-4, let-7 and other miRNA genes are conserved in mammals, and their potential functions in mammalian development are under active study.

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 normalmalignant 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 (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.

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Purpose MicroRNA (miRNA) expression profiles improve classification, diagnosis, and prognostic information of malignancies, including lung cancer. This study uncovered unique growthsuppressive 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 predictedmiRNA target was examined.ThesemiRNAs 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-1and 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 amechanism 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.

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-azaCdR 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. [Cancer Res 2009;69(24):9360-6]

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 ␣ (RAR␣). 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. PML͞RAR␣, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of retinoid response in APL is the proteasome-dependent PML͞RAR␣ degradation. UBE1L transfection triggered PML͞RAR␣ degradation, but transfection of a truncated UBE1L or E1 did not cause this degradation. A tight link was shown between UBE1L induction and PML͞RAR␣ degradation. Notably, retroviral expression of UBE1L rapidly induced apoptosis in NB4 APL cells, but not in cells lacking PML͞RAR␣ expression. UBE1L has been implicated directly in retinoid effects in APL and may be targeted for repression by PML͞ RAR␣. UBE1L is proposed as a direct pharmacological target that overcomes oncogenic effects of PML͞RAR␣ by triggering its degradation and signaling apoptosis in APL cells.

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.lung carcinogenesis ͉ lung cancer ͉ sonic hedgehog C yclin E binds to and activates cyclin-dependent kinase 2 (Cdk2) and promotes G 1 cell cycle transition (1, 2). Cyclin E overexpression shortens the G 1 cell cycle, alters S-phase progression, and causes chromosomal instability (CIN) (3,4). Cyclin E has oncogenic potential. Transgenic cyclin E expression in the mammary gland causes hyperplasia and carcinoma (5). Aberrant cyclin E expression occurs in premalignant lung lesions (6). Cyclin E expression has a negative prognostic impact in lung cancers (7-9). Tobacco carcinogens can transform immortalized human bronchial epithelial (HBE) cells and augment cyclin E expression (10). All-trans-retinoic acid (RA) chemoprevention represses cyclin E and associated Cdk2 kinase activity, causing G 1 arrest (10). This would permit repair of genomic DNA damage by carcinogens and was proposed as a chemoprevention mechanism (10, 11).Regulation of cyclin E is critical for cell cycle progression. Cyclin E accumulates late in G 1 and declines through S phase (1, 2). Cyclin E is regulated by the ubiquitin-proteasome pathway (12). The ubiquitin ligase Cullin 3 promotes ubiquitination of free cyclin E not bound to Cdk2 (13). Ubiquitination of Cdk2-bound cyclin E depends on phosphorylation of threonines Thr-62 and -380 as well as Ser-372 and -384 (14-16). Phosphorylation of these residues allows cyclin E to be recognized by Fbw7 (hCdc4) (15, 17, 18), a phosphoepitope-specific substrate recognition component of the Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase. Fbw7 mutations occur in malignancies and contribute to cell cycle deregulation (15,(18)(19)(20)(21). Mutations of ...