Intrinsic stress response pathways are frequently mobilized within tumor cells. The mediators of these adaptive mechanisms and how they contribute to carcinogenesis remain poorly understood. A striking example is heat shock factor 1 (HSF1), master transcriptional regulator of the heat shock response. Surprisingly, we found that loss of the tumor suppressor gene neurofibromatosis type 1 (Nf1) increased HSF1 levels and triggered its activation in mouse embryonic fibroblasts. As a consequence, Nf1 -/-cells acquired tolerance to proteotoxic stress. This activation of HSF1 depended on dysregulated MAPK signaling. HSF1, in turn, supported MAPK signaling. In mice, Hsf1 deficiency impeded NF1-associated carcinogenesis by attenuating oncogenic RAS/MAPK signaling. In cell lines from human malignant peripheral nerve sheath tumors (MPNSTs) driven by NF1 loss, HSF1 was overexpressed and activated, which was required for tumor cell viability. In surgical resections of human MPNSTs, HSF1 was overexpressed, translocated to the nucleus, and phosphorylated. These findings reveal a surprising biological consequence of NF1 deficiency: activation of HSF1 and ensuing addiction to this master regulator of the heat shock response. The loss of NF1 function engages an evolutionarily conserved cellular survival mechanism that ultimately impairs survival of the whole organism by facilitating carcinogenesis. IntroductionEvolutionarily conserved from yeasts to humans, the heat shock transcription factor heat shock factor 1 (HSF1) is activated by a broad range of stressors that extend far beyond heat, including heavy metals, UV radiation, hypoxia, desiccation, and acidosis (1). During activation, HSF1 undergoes phosphorylation and other posttranslational modifications, trimerization, and nuclear translocation. This results in rapid, high-affinity binding of HSF1 to consensus heat shock elements (HSEs) within the promoters of target genes (2). Such binding drives the induction or suppression of hundreds of genes in mammalian cells (3).The adaptive response unleashed by HSF1 activation is critical for maintaining homeostasis of the cell's proteome, mediated in large part by increased expression of classical heat shock proteins such as HSP27, HSP70, and HSP90 (4). However, the effect of HSF1 activation goes far beyond these chaperones. It helps coordinate a range of fundamental cellular processes that are important to the fitness of malignant cells, including cell cycle control, ribosome biogenesis, protein translation, and glucose metabolism (5, 6). As a result, HSF1 both facilitates initial oncogenic transformation and maintains the malignant phenotype of established cancer cell lines driven by a wide range of mutations. In mice and in cell culture, genetic ablation of Hsf1 expression potently impairs tumorigenesis and cellular transformation driven by oncogene activation or tumor suppressor loss (5). The importance of HSF1 in enabling malignancy has been demonstrated by other recent work as well

The existence of cancer stem cells (CSCs) or stem-like cancer cells (SLCCs) is regarded as the cause of tumor formation and recurrence. However, the origin of such cells remains controversial with two competing hypotheses: CSCs are either transformed from tissue adult stem cells or dedifferentiated from transformed progenitor cells. Compelling evidence has determined the chromosomal aneuploidy to be one of the hallmarks of cancer cells, indicating genome instability plays an important role in tumorigenesis, for which CSCs are believed to be the initiator. To gain direct evidence that genomic instability is involved in the induction of SLCCs, we utilized multiple approaches to enhance genomic instability and monitored the percentage of SLCC in cultured cancer cells. Using side population (SP) cells as a marker for SLCC in human nasopharyngeal carcinoma (NPC) and CD133 for human neuroblastoma cells, we found that DNA damage inducers, UV and mitomycin C were capable of increasing SP cells in NPC CNE-2 and neuroblastoma SKN-SH cells. Likewise, either overexpression of a key regulator of cell cycle, Mad2, or knock down of Aurora B, an important kinase in mitosis, or Cdh1, a key E3 ligase in cell cycle, resulted in a significant increase of SP cells in CNE-2. More interestingly, enrichment of SP cells was observed in recurrent tumor tissues as compared with the primary tumor in the same NPC patients. Our study thus suggested that, beside transformation of tissue stem cells leading to CSC generation, genomic instability could be another potential mechanism resulting in SLCC formation, especially at tumor recurrence stage.

Autophagy is essential for maintaining tissue homeostasis. Although adaptors have been demonstrated to facilitate the assembly of the Atg14L-Beclin 1-Vps34-Vps15 complex, which functions in autophagosome formation, it remains unknown whether the autophagy machinery actively recruits such adaptors. WD40-repeat proteins are a large, highly conserved family of adaptors implicated in various cellular activities. However, the role of WD40-repeat-only proteins, such as RACK1, in postnatal mammalian physiology remains unknown. Here, we report that hepatocyte-specific RACK1 deficiency leads to lipid accumulation in the liver, accompanied by impaired Atg14L-linked Vps34 activity and autophagy. Further exploration indicates that RACK1 participates in the formation of autophagosome biogenesis complex upon its phosphorylation by AMPK at Thr50. Thr50 phosphorylation of RACK1 enhances its direct binding to Vps15, Atg14L, and Beclin 1, thereby promoting the assembly of the autophagy-initiation complex. These observations provide insight into autophagy induction and establish a pivotal role for RACK1 in postnatal mammalian physiology.

The mechanisms of cotton fiber development and somatic embryogenesis have been explored systematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples, with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression results, normalization of real-time PCR data against one or several internal control genes is required, which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes, including 18S rRNA, Histone3, UBQ7, Actin, Cyclophilin, Gbpolyubiquitin-1 and Gbpolyubiquitin-2, in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene (arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quantification should be an efficient and convenient method for the fiber developmental series. The expression of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3, UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.cotton, fiber development, housekeeping genes, internal control, real-time PCR, somatic embryogenesis Quantitative real-time PCR (qRT-PCR) analysis has become a common, rapid and reliable method for the detection and quantification of mRNA transcription levels of the genes of interest. It is safer, and more sensitive and accurate than the Northern blot, and works with a minimal amount of starting material. A problem is that the experimental procedure could lead to severe misinterpretation of the results by different quantities and qualities of starting RNA, efficiencies of retrotranscription from RNA to cDNA, and efficiencies of PCR [1] , for which relative quantification is a commonly adopted strategy. The target concentration in each sample is calculated relative to another gene transcript, a housekeeping gene, and the result is expressed as a target/reference ratio. Housekeeping genes, such as 18S rRNA, ubiquitin, actin, tubulin, histone and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were chosen because of their known or expected roles in basic cellular processes (cell structure maintenance or primary metabolism) and the least variation in their expression under various conditions. However, some housekeeping genes vary considerably since biological systems are dynamic and constantly changing in response to their environment [2][3][4] . In order to choose the appropriate control genes, statistical algorithms, such as geNORM and BestKeeper have been

The present study explored the effect of long non-coding RNA-human ovarian cancer-specific transcript 2 (LncRNA-HOST2) on cell proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721. HCC tissues and adjacent normal tissues from 162 HCC patients were collected. The HCC cell lines were assigned into the control group (regular culture), negative control (NC) group (transfected with siRNA) and experimental group (transfected with Lnc-HOST2 siRNA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of LncRNA-HOST2. Cell proliferation was detected by CCK-8 and colony-forming assays, cell apoptosis by flow cytometry and cell migration by Scratch test. Transwell assay was used to evaluate cell migration and invasion abilities. LncRNA-HOST2 expression in the HCC tissues increased 2–10 times than that in the adjacent normal tissues. Compared with the HL-7702 cell line, LncRNA-HOST2 expression in HepG2, SMMC-7721 and Huh7 cell lines was all up-regulated, but the SMMC-7721 cell had the highest Lnc-HOST2 expression. The LncRNA-HOST2 expression in the experimental group was down-regulated as compared with the control and NC groups. In comparison with the control and NC groups, cloned cells reduced, cell apoptosis increased, clone-forming ability weakened and inhibitory rate of colony formation increased in the experimental group. The cells migrating and penetrating into the transwell chamber were fewer in the experimental group than those in the control and NC groups. The experimental group exhibited slow wound healing and decreased cell migration area after 48 h. These findings indicate that LncRNA-HOST2 can promote cell proliferation, migration and invasion and inhibit cell apoptosis in human HCC cell line SMMC-7721.