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Ferroptosis is a recently-defined tumor suppression mechanism, but the sensitivity of many tumorigenic cells to ferroptosis is limited by their deficient expression of acyl-CoA synthetase long-chain family member 4 (ACSL4). Here, we report the discovery of a photosensitizer, namely TPCI, which can evoke ACSL4-independent ferroptosis of cancer cells in photodynamic therapy. Through co-localization with 12-lipoxygenase (ALOX12) in multiple subcellular organelles, TPCI activates ALOX12 to generate lipid reactive oxygen species in large quantity and trigger cell ferroptosis. Intriguingly, confining TPCI exclusively in lysosomes switches the cell death from ferroptosis to apoptosis. More strikingly, the ferroptosis mediated by TPCI-induced ALOX12 activation does not require the participation of ACSL4. Therefore, our study identifies TPCI as the first ALOX12 activator to induce ferroptosis independent of ACSL4, which renders a viable therapeutic approach on the basis of distinct ferroptosis of cancer cells, regardless their ACSL4 expressions.

Failures to treat triple-negative breast cancer (TNBC) are mainly due to chemoresistance or radioresistance. We and others previously discovered that zinc finger E-box-binding homeobox 1 (ZEB1) is a massive driver causing these resistance. However, how to dynamically modulate the intrinsic expression of ZEB1 during cell cycle progression is elusive. Here integrated affinity purification combined with mass spectrometry and TCGA analysis identify a cell cycle-related E3 ubiquitin ligase, checkpoint with forkhead and ring finger domains (CHFR), as a key negative regulator of ZEB1 in TNBC. Functional studies reveal that CHFR associates with and decreases ZEB1 expression in a ubiquitinating-dependent manner and that CHFR represses fatty acid synthase (FASN) expression through ZEB1, leading to significant cell death of TNBC under chemotherapy. Intriguingly, a small-molecule inhibitor of HDAC under clinical trial, Trichostatin A (TSA), increases the expression of CHFR independent of histone acetylation, thereby destabilizes ZEB1 and sensitizes the resistant TNBC cells to conventional chemotherapy. In patients with basal-like breast cancers, CHFR levels significantly correlates with survival. These findings suggest the therapeutic potential for targeting CHFR-ZEB1 signaling in resistant malignant breast cancers.

Dysregulation of YAP localization and activity is associated with pathological conditions such as cancer. Although activation of the Hippo phosphorylation cascade is known to cause cytoplasmic retention and inactivation of YAP, emerging evidence suggests that YAP can be regulated in a Hippo-independent manner. Here, we report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCFSKP2 E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP’s nuclear localization, transcriptional activity, and growth-promoting function. Independently of Hippo signaling, mutation of YAP’s K63-linkage specific ubiquitination sites K321 and K497, depletion of SKP2, or overexpression of OTUD1 retains YAP in the cytoplasm and inhibits its activity. Conversely, overexpression of SKP2 or loss of OTUD1 leads to nuclear localization and activation of YAP. Altogether, our study sheds light on the ubiquitination-mediated, Hippo-independent regulation of YAP., Regulation of Yes-associated protein (YAP) through the Hippo pathway is well established, but its Hippo-independent regulation remains to be elucidated. Here, the authors show that non-proteolytic ubiquitination presents another means of YAP regulation, promoting its nuclear localization and activity.

Summary: Although EZH2 enzymatic inhibitors have shown antitumor effects in EZH2-mutated lymphoma and ARID1A-mutated ovarian cancer, many cancers do not respond because EZH2 can promote cancer independently of its histone methyltransferase activity. Here we identify ZRANB1 as the EZH2 deubiquitinase. ZRANB1 binds, deubiquitinates, and stabilizes EZH2. Depletion of ZRANB1 in breast cancer cells results in EZH2 destabilization and growth inhibition. Systemic delivery of ZRANB1 small interfering RNA (siRNA) leads to marked antitumor and antimetastatic effects in preclinical models of triple-negative breast cancer (TNBC). Intriguingly, a small-molecule inhibitor of ZRANB1 destabilizes EZH2 and inhibits the viability of TNBC cells. In patients with breast cancer, ZRANB1 levels correlate with EZH2 levels and poor survival. These findings suggest the therapeutic potential for targeting the EZH2 deubiquitinase ZRANB1. : Many cancer cells are sensitive to depletion of EZH2 but resistant to EZH2 inhibitors, due to EZH2’s enzyme-independent cancer-promoting function. Zhang et al. identify ZRANB1 as an EZH2 deubiquitinase and a potential anticancer target. Keywords: ZRANB1, EZH2, deubiquitinase, breast cancer

Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that promotes tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT) in carcinoma cells. EMT not only plays an important role in embryonic development and malignant progression, but is also implicated in cancer therapy resistance. It has been hypothesized that carcinoma cells that have undergone EMT acquire cancer stem cell properties including self-renewal, chemoresistance and radioresistance. However, our recent data indicate that ZEB1 regulates radioresistance in breast cancer cells through an EMT-independent mechanism. In this Perspective, we review different mechanisms by which ZEB1 regulates tumor progression and treatment resistance. Based on studies by us and others, we propose that it is specific EMT inducers like ZEB1, but not the epithelial or mesenchymal state itself, that dictate cancer stem cell properties.

Long non-coding RNAs (lncRNAs) have gained massive attention in recent years as a potentially new and crucial layer of gene regulation. LncRNAs are prevalently transcribed in the genome, but their roles in gene regulation and disease development are largely unknown. HOX antisense intergenic RNA (HOTAIR), a lncRNA located in the HOXC locus, has been shown to repress HOXD gene expression and promote breast cancer metastasis. Mechanistically, HOTAIR interacts with and recruits polycomb repressive complex 2 (PRC2) and regulates chromosome occupancy by EZH2 (a subunit of PRC2), which leads to histone H3 lysine 27 trimethylation of the HOXD locus. Moreover, HOTAIR is pervasively overexpressed in most human cancers compared with noncancerous adjacent tissues. This review summarizes the studies on the HOTAIR lncRNA over the past 6 years.

Although growing numbers of oncoproteins and pro-metastatic proteins have been extensively characterized, many of these tumor-promoting proteins are not good drug targets, which represents a major barrier to curing breast cancer and other cancers. There is a need, therefore, for alternative therapeutic approaches to destroying cancer-promoting proteins. The human genome encodes approximately 100 deubiquitinating enzymes (DUBs, also called deubiquitinases), which are amenable to pharmacologic inhibition by small molecules. By removing monoubiquitin or polyubiquitin chains from the target protein, DUBs can modulate the degradation, localization, activity, trafficking, and recycling of the substrate, thereby contributing substantially to the regulation of cancer proteins and pathways. Targeting certain DUBs may lead to destabilization or functional inactivation of some key oncoproteins or prometastatic proteins, including non-druggable ones, which will provide therapeutic benefits to cancer patients. In breast cancer, growing numbers of DUBs are found to be aberrantly expressed. Depending on their substrates, specific DUBs can either promote or suppress mammary tumors. In this article, we review the role and mechanisms of action of DUBs in breast cancer, and discuss the potential of targeting DUBs for cancer treatment.

Tumour cells associated with therapy resistance (radioresistance and drug resistance) are likely to give rise to local recurrence and distant metastatic relapse. Recent studies revealed microRNA (miRNA)-mediated regulation of metastasis and epithelial-mesenchymal transition; however, whether specific miRNAs regulate tumour radioresistance and can be exploited as radiosensitizing agents remains unclear. Here we find that miR-205 promotes radiosensitivity and is downregulated in radioresistant subpopulations of breast cancer cells, and that loss of miR-205 is highly associated with poor distant relapse-free survival in breast cancer patients. Notably, therapeutic delivery of miR-205 mimics via nanoliposomes can sensitize the tumour to radiation in a xenograft model. Mechanistically, radiation suppresses miR-205 expression through ataxia telangiectasia mutated (ATM) and zinc finger E-box binding homeobox 1 (ZEB1). Moreover, miR-205 inhibits DNA damage repair by targeting ZEB1 and the ubiquitin-conjugating enzyme Ubc13. These findings identify miR-205 as a radiosensitizing miRNA and reveal a new therapeutic strategy for radioresistant tumours.

Whether epithelial-mesenchymal transition (EMT) is always linked to increased tumorigenicity is controversial. Through microRNA (miRNA) expression profiling of mammary epithelial cells overexpressing Twist, Snail or ZEB1, we identified miR-100 as a novel EMT inducer. Surprisingly, miR-100 inhibits the tumorigenicity, motility and invasiveness of mammary tumor cells, and is commonly downregulated in human breast cancer due to hypermethylation of its host gene MIR100HG. The EMT-inducing and tumor-suppressing effects of miR-100 are mediated by distinct targets. While miR-100 downregulates E-cadherin by targeting SMARCA5, a regulator of CDH1 promoter methylation, this miRNA suppresses tumorigenesis, cell movement and invasion in vitro and in vivo through direct targeting of HOXA1, a gene that is both oncogenic and pro-invasive, leading to repression of multiple HOXA1 downstream targets involved in oncogenesis and invasiveness. These findings provide a proof-of-principle that EMT and tumorigenicity are not always associated and that certain EMT inducers can inhibit tumorigenesis, migration and invasion., Author Summary Induction of epithelial-mesenchymal transition (EMT) in epithelial tumor cells has been shown to enhance migration, invasion and cancer ‘stemness’. Here we demonstrate that a miRNA downregulated in human breast tumors, miR-100, can simultaneously induce EMT and inhibit tumorigenesis, migration and invasion through direct targeting of distinct genes. This is the first report of an EMT inducer that suppresses cell movement and tumor invasion, which indicates that EMT is not always associated with increased tumorigenesis, migration and invasion, and that all EMT inducers are not equal: while some of them can promote tumorigenicity, motility and invasiveness, others inhibit these properties owing to their ability to concurrently target both EMT-repressing genes and oncogenic/pro-invasive genes. These findings provide new insights into the complex roles of EMT inducers.

The tumour suppressor PTEN is frequently lost in human cancers. In addition to gene mutations and deletions, recent studies have revealed the importance of post-translational modifications, such as ubiquitylation, in the regulation of PTEN stability, activity and localization. However, the deubiquitylase that regulates PTEN polyubiquitylation and protein stability remains unknown. Here we screened a total of 30 deubiquitylating enzymes (DUBs) and identified five DUBs that physically associate with PTEN. One of them, USP13, stabilizes the PTEN protein through direct binding and deubiquitylation of PTEN. Loss of USP13 in breast cancer cells promotes AKT phosphorylation, cell proliferation, anchorage-independent growth, glycolysis and tumour growth through downregulation of PTEN. Conversely, overexpression of USP13 suppresses tumorigenesis and glycolysis in PTEN-positive but not PTEN-null breast cancer cells. Importantly, USP13 protein is downregulated in human breast tumours and correlates with PTEN protein levels. These findings identify USP13 as a tumour-suppressing protein that functions through deubiquitylation and stabilization of PTEN.

Objective: Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. P16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. Methods: HNSCC cells and xenografts were used. P16-overexpressing (HPV/p16-negative HN5 and UMSCC-1) and p16 shRNA knockdown (HPV/p16-positive UMSCC-47 and UCPI-SCC154) cells, TRIP12 shRNA or siRNA knockdown (HPV/p16-negative HN5 and Fadu) cells were generated. The effects of p16 or TRIP12 on HNSCC cell radiosensitivity were determined by clonogenic cell survival. The effects of p16 on tumor xenografts (HPV/p16-negative HN5 and HPV/p16-positive UMSCC-47) radioresponse were evaluated by tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction (PCR); and data from The Cancer Genome Atlas HNSCC project were analyzed. Results: P16 expression led to downregulation of TRIP12 protein both in vitro and in vivo via decreasing TRIP12 protein's half life, which in turn led to increased RNF168 levels and subsequently repressed DNA damage repair, represented by increased 53BP1 foci and neutral comet moment tails at 24 hours after irradiation. As a result, p16 expression enhanced radioresponsiveness both in vitro and in vivo. Inhibition of TRIP12 expression led to radiosensitization through repressed DNA damage repair, represented by decreased BRCA1 foci at 1 and 5 hours after irradiation; and increased neutral comet moment tails at 24 hours after irradiation. Furthermore, overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. Conclusions: The findings of our study reveal that p16 participates in radiosensitization through influencing DNA damage repair. P16 downregulates TRIP12 protein expression via post-translational regulation. Inhibition of TRIP12 sensitized HNSCC cells via prohibition of DNA damage repair. These findings support the rationale of blocking TRIP12 signaling to improve radiotherapy outcomes. Citation Format: Li Wang, Peijing Zhang, David Molkentine, Jessica Molkentine, Uma Raju, David Valdecanas, Ramesh Tailor, Howard Thames, Thomas Buchholz, Junjie Chen, Li Ma, Kathy Mason, Raymond Meyn, Heath D. Skinner. TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1661.

There is a pressing need to identify prognostic markers of metastatic disease and targets for treatment. Combining high-throughput RNA sequencing, functional characterization, mechanistic studies and clinical validation, we identify leukemia inhibitory factor receptor (LIFR) as a breast cancer metastasis suppressor downstream of the microRNA miR-9 and upstream of Hippo signaling. Restoring LIFR expression in highly malignant tumor cells suppresses metastasis by triggering a Hippo kinase cascade that leads to phosphorylation, cytoplasmic retention and functional inactivation of the transcriptional coactivator YES-associated protein (YAP). Conversely, loss of LIFR in nonmetastatic breast cancer cells induces migration, invasion and metastatic colonization through activation of YAP. LIFR is downregulated in human breast carcinomas and inversely correlates with metastasis. Notably, in approximately 1,000 nonmetastatic breast tumors, LIFR expression status correlated with metastasis-free, recurrence-free and overall survival outcomes in the patients. These findings identify LIFR as a metastasis suppressor that functions through the Hippo-YAP pathway and has significant prognostic power.

Author(s): Zhao, Jiang-Sha; Li, Wen-Jie; Ge, Di; Zhang, Pei-Jing; Li, Jing-Jing; Lu, Chun-Lai; Ji, Xiao-Dan; Guan, Dong-Xian; Gao, Hong; Xu, Li-Yan; Li, Eng-Ming; Soukiasian, Harmik; Koeffler, H Phillip; Wang, Xiao-Fan; Xie, Dong | Abstract: BackgroundEsophageal Squamous Cell Carcinoma (ESCC) is a major subtype of esophageal cancer causing significant morbility and mortality in Asia. Mechanism of initiation and progression of this disease is unclear. Tumor initiating cells (TICs) are the subpopulation of cells which have the ability to self-renew, as well as, to drive initiation and progression of cancer. Increasing evidence has shown that TICs exist in a variety of tumors. However, the identification and characterization of TICs in esophageal carcinoma has remained elusive.Methodology/principal findingsto identify TICs in ESCC, ESCC cell lines including two primary cells were used for screening suitable surface marker. Then colony formation assay, drug resistant assay and tumorigenicity assay in immune deficient mice were used to characterize TICs in ESCC. We found that just the CD44 expression correlated with tumorigenicity in ESCC cell lines. And then induced differentiation of ESCC cells by all-trans retinoic acid treatment led to decreased expression of CD44. The FACS isolated cell subpopulations with high CD44 expression showed increased colony formation and drug resistance in vitro, as well as significantly enhanced tumorigenicity in NOD/SICD mice, as compared to the low expressing CD44 ESCC cells.Conclusions/significanceour study has discovered a novel TIC surface marker, CD44, which can be utilized to enrich efficiently the TICs in ESCC. These findings will be useful for further studies of these cells and exploring therapeutic approaches.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent inducer of tumor cell apoptosis, but concerns of considerable liver toxicity limit its uses in human cancer therapy. Here, we show that i.v. injected Escherichia coli DH5alpha (E. coli DH5alpha) specifically replicates in solid tumors and metastases in live animals. E. coli DH5alpha does not enter tumor cells and suits for being the vector for soluble TRAIL (sTRAIL), which induces apoptosis by activating cell-surface death receptors. With the high 'tumor-targeting' nature, we demonstrate that intratumoral (i.t.) and intravenous injection of sTRAIL-expressing E. coli DH5alpha results in the tumor-targeted release of biologically active molecules, which leads to a dramatic reduction in the tumor growth rate and the prolonged survival of tumor-bearing mice. TRAIL delivery by E. coli DH5alpha did not cause any detectable toxicity to any organs, suggesting that E. coli DH5alpha-delivered sTRAIL protein therapy may provide a feasible and effective form of treatment for solid tumors.

DNA damage response (DDR) acts as a tumorigenesis barrier, and any defects in the DDR machinery may lead to cancer. SOX4 expression is elevated in many types of tumors; however, its role in DDR is still largely unknown. Here, we show that SOX4, a new DNA damage sensor, is required for the activation of p53 tumor suppressor in response to DNA damage. Notably, SOX4 interacts with and stabilizes p53 protein by blocking Mdm2-mediated p53 ubiquitination and degradation. Furthermore, SOX4 enhances p53 acetylation by interacting with p300/CBP and facilitating p300/CBP/p53 complex formation. In concert with these results, SOX4 promotes cell cycle arrest and apoptosis, and it inhibits tumorigenesis in a p53-dependent manner. Therefore, these findings highlight SOX4 as a potential key factor in regulating DDR-associated cancer.

Human papillomavirus (HPV) type 16 is a major cause of oropharyngeal carcinoma (OPC). P16INK4A has been suggested to be a reliable surrogate marker of HPV-associated OPC with 100% sensitivity and about 80% specificity. Increasing data showed tumor HPV positivity or p16 expression was strongly associated with significantly better prognosis in patients with OPC. As a tumor suppressor gene, p16 has biological functions including regulation of cell cycle progression at the G1/S boundary, angiogenesis, cell senescence, tumor invasion, cell spreading, apoptosis and anoikis. The present study was undertaken to assess the role of p16 in regulating tumor radioresponse and the underlying mechanisms in OPC cell lines. OPC cell lines HN-5 (HPV and p16 negative) and UMSCC-47 (HPV and p16 positive) were used. P16 overexpressing HN-5 cells and shRNA p16 knockdown UMSCC-47 cells were generated using lentivirus vectors. Treatment endpoint was clonogenic cell survival (CSA) determined 10-12 days (for HN5) or 17-20 days (for UMSCC-47) after exposing the cells to 2-10 Gy single doses of γ-radiation (IR). Compared with the control (scramble) cells, p16-overexpressing HN5 cells had significantly higher radiosensitivity (by a factor of 1.56 at 0.1 cell survival fraction); whereas, p16-knockingdown UMSCC-47 cells had less radiosensitivity (by a factor of 1.23 at 0.1 cell survival fraction). Overexpressing P16 in HN-5 cells significantly prolonged the presence of radiation-induced double-strand breaks detected on the basis of 53BP1 foci at 24h after 4 Gy IR. To directly gauge damaged DNA, an alkaline comet assay to detect both single- and double-strand DNA breaks was performed. P16 overexpressing HN-5 cells exhibited a 2.09-fold increase in the comet ‘tail moment’ 48 hours after IR. This finding was supported by increased expression of 53BP1 analyzed by Western blot. In conclusion, other than being a robust surrogate marker for tumor control and survival outcome, our findings demonstrated that p16 also functions as a potent radiation sensitizer. The major underlying mechanism of p16 regulating radiosensitivity is by inhibition of DNA damage repair. Citation Format: Li Wang, Peijing Zhang, David P Molkentine, Hailong Piao, Chunyan Chen, Jessica M Molkentine, Jinsong Zhang, David R Valdecanas, Heath Skinner, Thomas A Buchholz, Junjie Chen, Li Ma, Kathy A Mason, Kie-kian Ang, Raymond E Meyn. P16INK4A, a surrogate marker of HPV infection and prognosis for head and neck cancer, delays DNA damage repair and enhances radiation response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4895. doi:10.1158/1538-7445.AM2014-4895