g S. Chan

Autophagy Is Required for Glucose Homeostasis and Lung Tumor Maintenance

UNLABELLED Macroautophagy (autophagy hereafter) recycles intracellular components to sustain mitochondrial metabolism that promotes the growth, stress tolerance, and malignancy of lung cancers, suggesting that autophagy inhibition may have antitumor activity. To assess the functional significance of autophagy in both normal and tumor tissue, we conditionally deleted the essential autophagy gene, autophagy related 7 (Atg7), throughout adult mice. Here, we report that systemic ATG7 ablation caused susceptibility to infection and neurodegeneration that limited survival to 2 to 3 months. Moreover, upon fasting, autophagy-deficient mice suffered fatal hypoglycemia. Prior autophagy ablation did not alter the efficiency of non-small cell lung cancer (NSCLC) initiation by activation of oncogenic Kras(G12D) and deletion of the Trp53 tumor suppressor. Acute autophagy ablation in mice with preexisting NSCLC, however, blocked tumor growth, promoted tumor cell death, and generated more benign disease (oncocytomas). This antitumor activity occurred before destruction of normal tissues, suggesting that acute autophagy inhibition may be therapeutically beneficial in cancer. SIGNIFICANCE We systemically ablated cellular self-cannibalization by autophagy in adult mice and determined that it is dispensable for short-term survival, but required to prevent fatal hypoglycemia and cachexia during fasting, delineating a new role for autophagy in metabolism. Importantly, acute, systemic autophagy ablation was selectively destructive to established tumors compared with normal tissues, thereby providing the preclinical evidence that strategies to inhibit autophagy may be therapeutically advantageous for RAS-driven cancers.

Autophagy, Metabolism, and Cancer.

Macroautophagy (autophagy hereafter) captures intracellular proteins and organelles and degrades them in lysosomes. The degradation breakdown products are released from lysosomes and recycled into metabolic and biosynthetic pathways. Basal autophagy provides protein and organelle quality control by eliminating damaged cellular components. Starvation-induced autophagy recycles intracellular components into metabolic pathways to sustain mitochondrial metabolic function and energy homeostasis. Recycling by autophagy is essential for yeast and mammals to survive starvation through intracellular nutrient scavenging. Autophagy suppresses degenerative diseases and has a context-dependent role in cancer. In some models, cancer initiation is suppressed by autophagy. By preventing the toxic accumulation of damaged protein and organelles, particularly mitochondria, autophagy limits oxidative stress, chronic tissue damage, and oncogenic signaling, which suppresses cancer initiation. This suggests a role for autophagy stimulation in cancer prevention, although the role of autophagy in the suppression of human cancer is unclear. In contrast, some cancers induce autophagy and are dependent on autophagy for survival. Much in the way that autophagy promotes survival in starvation, cancers can use autophagy-mediated recycling to maintain mitochondrial function and energy homeostasis to meet the elevated metabolic demand of growth and proliferation. Thus, autophagy inhibition may be beneficial for cancer therapy. Moreover, tumors are more autophagy-dependent than normal tissues, suggesting that there is a therapeutic window. Despite these insights, many important unanswered questions remain about the exact mechanisms of autophagy-mediated cancer suppression and promotion, how relevant these observations are to humans, and whether the autophagy pathway can be modulated therapeutically in cancer. Clin Cancer Res; 21(22); 5037–46. ©2015 AACR. See all articles in this CCR Focus section, “Cell Death and Cancer Therapy.”

Mutational Landscape of the Essential Autophagy Gene BECN1 in Human Cancers

Evidence suggests that the catabolic process of macroautophagy (autophagy hereafter) can either suppress or promote cancer. The essential autophagy gene ATG6/BECN1 encoding the Beclin1 protein has been implicated as a haploinsufficient tumor suppressor in breast, ovarian, and prostate cancers. The proximity of BECN1 to the known breast and ovarian tumor suppressor breast cancer 1, early onset, BRCA1, on chromosome 17q21, has made this determination equivocal. Here, the mutational status of BECN1 was assessed in human tumor sequencing data from The Cancer Genome Atlas (TCGA) and other databases. Large deletions encompassing both BRCA1 and BECN1, and deletions of only BRCA1 but not BECN1, were found in breast and ovarian cancers, consistent with BRCA1 loss being a primary driver mutation in these cancers. Furthermore, there was no evidence for BECN1 mutation or loss in any other cancer, casting doubt on whether BECN1 is a tumor suppressor in most human cancers. Implications: Contrary to previous reports, BECN1 is not significantly mutated in human cancer and not a tumor-suppressor gene, as originally thought. Visual Overview: http://mcr.aacrjournals.org/content/early/2014/04/01/1541-7786.MCR-13-0614/F1.large.jpg. Mol Cancer Res; 12(4); 485–90. ©2014 AACR.

Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells

Autophagy degrades and is thought to recycle proteins, other macromolecules, and organelles. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes malignancy. Autophagy-deficient tumor-derived cell lines are respiration-impaired and starvation-sensitive. However, to what extent their sensitivity to starvation arises from defective mitochondria or an impaired supply of metabolic substrates remains unclear. Here, we sequenced the mitochondrial genomes of wild-type or autophagy-deficient (Atg7(-/-)) Kras-driven lung tumors. Although Atg7 deletion resulted in increased mitochondrial mutations, there were too few nonsynonymous mutations to cause generalized mitochondrial dysfunction. In contrast, pulse-chase studies with isotope-labeled nutrients revealed impaired mitochondrial substrate supply during starvation of the autophagy-deficient cells. This was associated with increased reactive oxygen species (ROS), lower energy charge, and a dramatic drop in total nucleotide pools. While starvation survival of the autophagy-deficient cells was not rescued by the general antioxidant N-acetyl-cysteine, it was fully rescued by glutamine or glutamate (both amino acids that feed the TCA cycle and nucleotide synthesis) or nucleosides. Thus, maintenance of nucleotide pools is a critical challenge for starving Kras-driven tumor cells. By providing bioenergetic and biosynthetic substrates, autophagy supports nucleotide pools and thereby starvation survival.

The Genomic Landscape of Renal Oncocytoma Identifies a Metabolic Barrier to Tumorigenesis

SUMMARY Oncocytomas are predominantly benign neoplasms possessing pathogenic mitochondrial mutations and accumulation of respiration-defective mitochondria, characteristics of unknown significance. Using exome and transcriptome sequencing, we identified two main subtypes of renal oncocytoma. Type 1 is diploid with CCND1 rearrangements, whereas type 2 is aneuploid with recurrent loss of chromosome 1, X or Y, and/or 14 and 21, which may proceed to more aggressive eosinophilic chromophobe renal cell carcinoma (ChRCC). Oncocytomas activate 5′ adenosine monophosphate-activated protein kinase (AMPK) and Tp53 (p53) and display disruption of Golgi and autophagy/lysosome trafficking, events attributed to defective mitochondrial function. This suggests that the genetic defects in mitochondria activate a metabolic checkpoint, producing autophagy impairment and mitochondrial accumulation that limit tumor progression, revealing a novel tumor-suppressive mechanism for mitochondrial inhibition with metformin. Alleviation of this metabolic checkpoint in type 2 by p53 mutations may allow progression to eosinophilic ChRCC, indicating that they represent higher risk.

CCCTC-Binding Factor Confines the Distal Action of Estrogen Receptor

Distal enhancers have recently emerged as a common mode of gene regulation for several transcription factors, including estrogen and androgen receptors, the two key regulators of breast and prostate cancer major subtypes. Despite the rapid success in genome-wide annotation of estrogen receptor-alpha (ERalpha) binding sites in cell lines, the precise mechanism governing the gene-to-enhancer association is still unknown and no quantitative model that can predict the estrogen responsiveness of genes has been hitherto proposed. This article presents an integrative genomics approach to construct a predictive model that can explain more than 70% of estrogen-induced expression profiles. The proposed method combines a recent map of the insulator protein CCCTC-binding factor (CTCF) with previous ER location studies and expression profiling in the presence of the translation inhibitor cycloheximide, providing evidence that CTCF partitions the human genome into distinct ER-regulatory blocks. It is shown that estrogen-responsive genes with a decreased transcription level (down-regulated genes) have a markedly different relative distribution of ER binding sites compared with those with an increased transcription level (up-regulated genes). Finally, Bayesian belief networks are constructed to quantify the effects of ER-binding distance from genes as well as the insulating effects of CTCF on the estrogen responsiveness of genes. This work thus represents a stride toward understanding and predicting the distal activities of steroid hormone nuclear receptors.

Clinical Actionability of Comprehensive Genomic Profiling for Management of Rare or Refractory Cancers

To study the frequency with which targeted tumor sequencing results will lead to implemented change in care, this study assessed tumors from 100 patients for utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. Comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations.

Involvement of tumor suppressors PTEN and p53 in the formation of multiple subtypes of liposarcoma

Liposarcoma (LPS) is a type of soft tissue sarcoma that mostly occurs in adults, and in humans is characterized by amplifications of MDM2 and CDK4. The molecular pathogenesis of this malignancy is still poorly understood and, therefore, we developed a mouse model with conditional inactivation of PTEN and p53 to investigate these pathways in the progression of the disease. We show that deletion of these two tumor suppressors cooperate in the formation of multiple subtypes of LPS (from well-differentiated LPS to pleomorphic LPS). In addition, progression of the tumors is further characterized by the expression of D cyclins and CDK4/6, which allow for continued cell division. Microarray analysis also revealed novel genes that are differentially expressed between different subtypes of LPS, which could aid in understanding the disease and to unravel potential new therapeutic targets.

Whole-genome sequencing analysis of phenotypic heterogeneity and anticipation in Li-Fraumeni cancer predisposition syndrome

Significance Germ-line mutation in the tumor suppressor TP53 causes Li–Fraumeni syndrome (LFS), a complex predisposition to multiple cancers. Types of cancers and ages at diagnosis vary among subjects and families, with apparent genetic anticipation: i.e., earlier cancer onset with successive generations. It has been proposed that anticipation is caused by accumulation of copy-number variations (CNV) in a context of TP53 haploinsufficiency. Using genome/exome sequencing, we found no evidence of increased rates of CNVs in two successive generations of TP53 mutation carriers and in successive generations of Trp53-deficient mice. We propose a stochastic model called “genetic regression” to explain apparent anticipation in LFS, caused by segregation of rare SNP and de novo mutations rather than by cumulative DNA damage. The Li–Fraumeni syndrome (LFS) and its variant form (LFL) is a familial predisposition to multiple forms of childhood, adolescent, and adult cancers associated with germ-line mutation in the TP53 tumor suppressor gene. Individual disparities in tumor patterns are compounded by acceleration of cancer onset with successive generations. It has been suggested that this apparent anticipation pattern may result from germ-line genomic instability in TP53 mutation carriers, causing increased DNA copy-number variations (CNVs) with successive generations. To address the genetic basis of phenotypic disparities of LFS/LFL, we performed whole-genome sequencing (WGS) of 13 subjects from two generations of an LFS kindred. Neither de novo CNV nor significant difference in total CNV was detected in relation with successive generations or with age at cancer onset. These observations were consistent with an experimental mouse model system showing that trp53 deficiency in the germ line of father or mother did not increase CNV occurrence in the offspring. On the other hand, individual records on 1,771 TP53 mutation carriers from 294 pedigrees were compiled to assess genetic anticipation patterns (International Agency for Research on Cancer TP53 database). No strictly defined anticipation pattern was observed. Rather, in multigeneration families, cancer onset was delayed in older compared with recent generations. These observations support an alternative model for apparent anticipation in which rare variants from noncarrier parents may attenuate constitutive resistance to tumorigenesis in the offspring of TP53 mutation carriers with late cancer onset.

microRNA-1827 represses MDM2 to positively regulate tumor suppressor p53 and suppress tumorigenesis

The tumor suppressor p53 plays a central role in tumor prevention. The E3 ubiquitin ligase MDM2 is the most critical negative regulator of p53, which binds to p53 and degrades p53 through ubiquitation. MDM2 itself is a transcriptional target of p53, and therefore, MDM2 forms a negative feedback loop with p53 to tightly regulate p53 levels and function. microRNAs (miRNAs) play a key role in regulation of gene expression. miRNA dysregulation plays an important role in tumorigenesis. In this study, we found that miRNA miR-1827 is a novel miRNA that targets MDM2 through binding to the 3′-UTR of MDM2 mRNA. miR-1827 negatively regulates MDM2, which in turn increases p53 protein levels to increase transcriptional activity of p53 and enhance p53-mediated stress responses, including apoptosis and senescence. Overexpression of miR-1827 suppresses the growth of xenograft colorectal tumors, whereas the miR-1827 inhibitor promotes tumor growth in mice in a largely p53-dependent manner. miR-1827 is frequently down-regulated in human colorectal cancer. Decreased miR-1827 expression is associated with high MDM2 expression and poor prognosis in colorectal cancer. In summary, our results reveal that miR-1827 is a novel miRNA that regulates p53 through targeting MDM2, and highlight an important role and the underlying mechanism of miR-1827 in tumor suppression.