The selective autophagy substrate p62 serves as a molecular link between autophagy and cancer. Suppression of autophagy causes p62 accumulation and thereby contributes to tumorigenesis. Here we demonstrate that autophagy deficiency promotes cell proliferation and migration through p62-dependent stabilization of the oncogenic transcription factor Twist1. p62 binds to Twist1 and inhibits degradation of Twist1. In mice, p62 up-regulation promotes tumor cell growth and metastasis in a Twist1-dependent manner. Our findings demonstrate that Twist1 is a key downstream effector of p62 in regulation of cell proliferation and migration and suggest that targeting p62-mediated Twist1 stabilization is a promising therapeutic strategy for prevention and treatment of cancer.acroautophagy (hereafter autophagy) is a catabolic process by which cellular proteins, cytoplasm, and organelles are captured and targeted for proteolytic degradation in lysosomes (1, 2). Autophagy dysfunction is associated with multiple human diseases, such as neurodegeneration, microbial infection, metabolic diseases, cardiovascular diseases, aging, and cancer (2-4). The multidomain protein p62/A170/SQSTM1 (hereafter referred to as "p62") has been shown to be both a selective autophagy substrate and an autophagy adaptor protein that acts as a link between ubiquitination and autophagy (5, 6). Several studies have demonstrated the oncogenic role of p62 in tumor formation and/ or progression (7, 8) through regulating NF-kappaB (9, 10) and NRF2 (11-13). Furthermore, Ras induces p62 expression in tumorigenesis (10). However, much remains to be elucidated with regard to its function and interaction with other critical cellular pathways.The transcription factor Twist1 is a core regulator in both early embryonic morphogenesis and cancer development and metastasis (14-17). It induces loss of epithelial (E)-cadherin-mediated cellcell adhesion and facilitates the epithelial-mesenchymal transition (EMT) (16), and it promotes cell proliferation (17). Twist1 is a basic helix-loop-helix (bHLH) protein and is structurally unrelated to other EMT factors including Slug and Snail. Recent studies have shown that Twist1 is a labile protein regulated by the ubiquitin-proteasome system through the F-box protein and E3 ubiquitin ligase Ppa (18). Despite these advances, the regulatory and functional role of Twist1 remains poorly understood. Here we demonstrate that p62 stabilizes Twist1 protein to increase cell proliferation and migration in vitro and in mice. Results Autophagy Deficiency Decreases E-Cadherin Expression and PromotesCell Migration, Invasion, and Proliferation. Two distinctive hallmarks of autophagy are the conversion of light chain 3-I (LC3-I) to LC3-II and the selective degradation of p62 (19,20). Multiple mammalian homologs of products of the autophagy-related genes (Atg) originally identified in yeast have been identified (3). Compared with wild-type (WT) mouse embryonic fibroblast (MEF) cells, cells with Atg3, Atg5, Atg9, and Atg12 knockout (KO) blocked the co...

Key Points• Del(18p), together with del(17p)/TP53 mutations, is present at a high frequency before ibrutinib treatment.• BTK mutations drive ibrutinib relapse, but del(17p)/TP53 mutations may be dispensable.Ibrutinib has generated remarkable responses in patients with chronic lymphocytic leukemia (CLL), including those with an unfavorable cytogenetic profile. However, patients develop resistance, with poor outcomes and no established treatment options. Mutations in BTK and PLCG2 have emerged as main mechanisms of drug resistance, but not all patients carry these mutations. Further understanding of mechanisms of resistance is urgently needed and will support rational development of new therapeutic strategies. To that end, we characterized the genomic profiles of serial samples from 9 patients with ibrutinib-relapsed disease, including 6 who had Richter transformation. Mutations, indels, copy-number aberrations, and loss of heterozygosity were assessed using next-generation sequencing and single-nucleotide polymorphism array. We found that 18p deletion (del(18p)), together with del(17p)/TP53 mutations, was present in 5 of 9 patients before ibrutinib therapy. In addition to BTK C481 , we identified BTK T316A, a structurally novel mutation located in the SH2 domain of BTK. Minor BTK clones with low allele frequencies were captured in addition to major BTK clones. Although TP53 loss predisposes patients for relapse, clone size of TP53 loss may diminish during disease progression while mutant BTK clone expands. In patients who had Richter transformation, we found that the transformed cells were clonal descendants of circulating leukemia cells but continued to undergo evolution and drifts.Surprisingly, transformed lymphoma cells in tissue may acquire a different BTK mutation from that in the CLL leukemia cells. Collectively, these results provide insights into clonal evolution underlying ibrutinib relapse and prompt further investigation on genomic abnormalities that have clinical application potential.

Disruption of the nucleotide excision repair (NER) pathway by mutations can cause xeroderma pigmentosum, a syndrome predisposing affected individuals to development of skin cancer. The xeroderma pigmentosum C (XPC) protein is essential for initiating global genome NER by recognizing the DNA lesion and recruiting downstream factors. Here we show that inhibition of the deacetylase and longevity factor SIRT1 impairs global genome NER through suppressing the transcription of XPC in a SIRT1 deacetylase-dependent manner. SIRT1 enhances XPC expression by reducing AKTdependent nuclear localization of the transcription repressor of XPC. Finally, we show that SIRT1 levels are significantly reduced in human skin tumors from Caucasian patients, a population at highest risk. These findings suggest that SIRT1 acts as a tumor suppressor through its role in DNA repair.ucleotide excision repair (NER) is a versatile DNA repair pathway that eliminates a wide variety of helix-distorting base lesions, including UV radiation-induced cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) (1, 2), as well as bulky adducts induced by numerous chemical compounds. Defects in NER by mutations cause the autosomal recessive xeroderma pigmentosum (XP) and Cockayne syndromes (3-5). XP patients are clinically characterized by cutaneous sensitivity to sunlight exposure and a predisposition to skin cancer. Seven NER-deficient genetic complementation groups of XP (XP-A to -G) have been identified, and all of the corresponding genes have now been cloned (3-5).Mammalian NER consists of two distinct subpathways: global genome NER (GG-NER), which operates throughout the genome, and transcription-coupled NER (TC-NER), which specifically removes lesions on the transcribed DNA strand of active genes. A major difference between these two pathways appears to lie in the strategies for detecting damaged bases. Accumulating evidence indicates that the XP group C (XPC) protein plays an essential role in GG-NER-specific damage recognition (6-8). Although biochemical and genetic analyses have characterized the function of XPC in considerable detail, much remains to be elucidated with regard to its regulation and interaction with other critical cellular pathways.Sirtuin 1 (SIRT1), a mammalian counterpart of the yeast silent information regulator 2 (Sir2) and a proto member of the sirtuin family, is an NAD-dependent longevity-promoting deacetylase. SIRT1 is crucial for cell survival, metabolism, senescence, and stress response in several cell types and tissues (9-13). Both histone and nonhistone targets of SIRT1 have been identified, including FOXO, p53, and PPARγ (10,12,14). SIRT1 has been implicated as an important player in cancer. However, it remains unclear whether SIRT1 serves as a tumor suppressor or a tumor promoter (13-16). SIRT1 expression is relatively higher in many malignancies, including colon, breast, prostate, and skin cancers and leukemia, as compared with their corresponding normal tissues (17)(18)(19)(20)(21)(22). But the specific c...

Skin cancer is the most common cancer in the U.S., while DNA-damaging UVB radiation from the sun remains the major environmental risk factor. Reducing skin cancer incidence is becoming an urgent issue. The energy-sensing enzyme 5’-AMP-activated protein kinase (AMPK) plays a key role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise and changes in fuel availability. However, the role AMPK in the response of skin cells to UVB damage and in skin cancer prevention remains unknown. Here we show that AMPK activation is reduced in human and mouse squamous cell carcinoma as compared with normal skin, and by UVB irradiation, suggesting that AMPK is a tumor suppressor. At the molecular level, AMPK deletion reduced the expression of the DNA repair protein xeroderma pigmentosum C (XPC) and UVB-induced DNA repair. AMPK activation by its activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin (N’,N’-dimethylbiguanide), the most widely used anti-diabetic drug, increased the expression of XPC expression and UVB-induced DNA repair in mouse skin, normal human epidermal keratinocytes, and AMPK wild-type cells but not in AMPK deficient cells, indicating an AMPK-dependent mechanism. Topical treatment with AICAR and metformin not only delayed onset of UVB-induced skin tumorigenesis but also reduced tumor multiplicity. Furthermore, AMPK deletion increased ERK activation and cell proliferation, while AICAR and metformin inhibited ERK activation and cell proliferation in keratinocytes, mouse skin, AMPK wild-type and AMPK deficient cells, suggesting an AMPK-independent mechanism. Finally, in UVB-damaged tumor-bearing mice, both topical and systemic metformin prevented the formation of new tumors and suppressed growth of established tumors. Our findings not only suggest that AMPK is a tumor suppressor in the skin by promoting DNA repair and controlling cell proliferation, but also demonstrate previously unknown mechanisms by which the AMPK activators prevent UVB-induced skin tumorigenesis.

The ability of DNA repair in a cell is vital to its genomic integrity and thus to the normal functioning of an organism. Phosphatase and tensin homolog (PTEN) is a well-established tumor suppressor gene that induces apoptosis and controls cell growth by inhibiting the PI3K/AKT pathway. In various human cancers, PTEN is frequently found to be mutated, deleted, or epigenetically silenced. Recent new findings have demonstrated that PTEN also plays a critical role in DNA damage repair and DNA damage response. This review summarizes the recent progress in the function of PTEN in DNA damage repair, especially in double strand break repair and nucleotide excision repair. In addition, we will discuss the role of PTEN in DNA damage response through its interaction with the Chk1 and p53 pathways. We will focus on the newly discovered mechanisms and the potential implications in cancer prevention and therapeutic intervention.

SIRT6 is a SIR2 family member that regulates multiple molecular pathways involved in metabolism, genomic stability and aging. It has been proposed previously that SIRT6 is a tumor suppressor in cancer. Here we challenge this concept by presenting evidence that skin-specific deletion of SIRT6 in the mouse inhibits skin tumorigenesis. SIRT6 promoted expression of COX-2 by repressing AMPK signaling, thereby increasing cell proliferation and survival and in the skin epidermis. SIRT6 expression in skin keratinocytes was increased by exposure to UVB light through activation of the AKT pathway. Clinically, we found that SIRT6 was upregulated in human skin squamous cell carcinoma. Taken together, our results provide evidence that SIRT6 functions an oncogene in the epidermis and suggest greater complexity to its role in epithelial carcinogenesis.

Non-melanoma skin cancer is the most common cancer in the U.S., where DNA-damaging UVB radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared to skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCC, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin, through its ability to positively regulate XPC-dependent GG-NER following DNA damage.

Background:The role of autophagy in genotoxic stress remains poorly understood. Results: Autophagy deficiency sensitizes cells to UVB-induced apoptosis through p62-dependent p38 activation. Conclusion: Inhibition of autophagy promotes apoptosis following UVB damage by increasing p38 activation. Significance: These findings identify the vital role of autophagy in cell survival under genotoxic stress and suggest the function of autophagy in cancer pathogenesis.