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Figure S8. Effect of the inhibition of multiple components of the PI3K/mTOR pathway in head and neck squamous cell carcinoma (HNSCC) cell lines. A. NOTCH1MUT cells (red text) and NOTCH1WT cells (black text) were treated with increasing concentrations (0-200 nM, 2-fold dilution) of GSK2126458 (a dual PI3K/mTOR inhibitor), BAY806946 (a pan-PI3K inhibitor), rapamycin and ridaforolimus (two mTOR inhibitors), and MK2206 (an AKT inhibitor) for 72 hours, and cell viability was assessed using the CellTiter-Glo assay. B. The protein p-S6 (S240/244) bands in Figure 2D were quantified using ImageJ software and normalized to β-actin expression, and the fold change from the control was calculated. Data are the means {plus minus} standard errors for three cell lines. *p

Table S3. Spearman correlation of protein expression to drug sensitivity based on 70% inhibitory concentration (IC70) or area under the curve (AUC)

Purpose:Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression.Results:PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1MUT but not NOTCH1WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1WT HNSCC but had little effect as single agents.Conclusions:Our findings suggest that NOTCH1MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1WT HNSCC to PI3K/mTOR pathway inhibitors.

Purpose: Ulcerative colitis and colitis-associated colorectal cancer (CAC) is a serious health issue, but etiopathological factors remain unclear. Aldo-keto reductase 1B10 (AKR1B10) is specifically expressed in the colonic epithelium, but downregulated in colorectal cancer. This study was aimed to investigate the etiopathogenic role of AKR1B10 in ulcerative colitis and CAC.Experimental Design: Ulcerative colitis and CAC biopsies (paraffin-embedded sections) and frozen tissues were collected to examine AKR1B10 expression. Aldo-keto reductase 1B8 (the ortholog of human AKR1B10) knockout (AKR1B8−/−) mice were produced to estimate its role in the susceptibility and severity of chronic colitis and associated dysplastic lesions, induced by dextran sulfate sodium (DSS) at a low dose (2%). Genome-wide exome sequencing was used to profile DNA damage in DSS-induced colitis and tumors.Results: AKR1B10 expression was markedly diminished in over 90% of ulcerative colitis and CAC tissues. AKR1B8 deficiency led to reduced lipid synthesis from butyrate and diminished proliferation of colonic epithelial cells. The DSS-treated AKR1B8−/− mice demonstrated impaired injury repair of colonic epithelium and more severe bleeding, inflammation, and ulceration. These AKR1B8−/− mice had more severe oxidative stress and DNA damage, and dysplasias were more frequent and at a higher grade in the AKR1B8−/− mice than in wild-type mice. Palpable masses were seen in the AKR1B8−/− mice only, not in wild-type.Conclusions: AKR1B8 is a critical protein in the proliferation and injury repair of the colonic epithelium and in the pathogenesis of ulcerative colitis and CAC, being a new etiopathogenic factor of these diseases. Clin Cancer Res; 21(6); 1466–76. ©2014 AACR.

Supplemental Figures S1-6, Tables S1-7. Figure S1. Targeted disruption of AKR1B8 gene. Figure S2. AKR1B and AKR1C subfamily expression. Figure S3. Deficiency of proliferation and self-renewal of colonic crypt cells in AKR1B8 -/- mice. Figure S4. DSS treatment schedules. Figure S5. Spleen size and histological inflammation of AKR1B8 -/- mice treated with 2% DSS. Figure S6. Colitis-associated tumorigenesis in AKR1B8 -/- mice. Supplemental Table S1. Coverage summary of Exome sequencing. Supplemental Table S2. Variations detected throughout the mouse Exome sequencing. Supplemental Table S3. Oncogenes and tumor suppressor genes mutated in AKR1B8 -/- colitis and tumors only, but not in wild type colitis. Supplemental Table S4. Disease Activity Index. Supplemental Table S5. Histopathological Inflammation Score. Supplemental Table S6. Definition of histopathology. Supplemental Table S7. Gene specific primers.

Long non-coding RNAs (lncRNAs) are non-coding RNAs longer than 200 nucleotides that function as regulatory factors in many human diseases, including cancer. However, majority of lncRNAs remain to be characterized. In this study, we characterized a novel lncRNA transcript, named UNC5B antisense RNA1 (UASR1). UASR1 is 647bp in length consisting of two exons. This lncRNA is an antisense of intron 1 of unc-5 netrin receptor B (UNC5B) gene. In breast cancer tissues, UASR1 was upregulated. Ectopic expression of UASR1 promoted proliferation and clonogenic growth of breast cancer cells MCF7 and MDA-MB-231. The migration of these cells also increased as demonstrated by wound healing and transwell assays. In contrast, silencing of UASR1 suppressed cell proliferation and migration. Further studies showed that UASR1 activated AKT and AKT-mediated mTOR signaling pathway to stimulate cell proliferation and growth. In these cells, active pAKT, pTSC2, p4EBP1 and pp70S6K were increased. Taken together, our data suggest that UASR1 plays an oncogenic role in breast cancer cells through activation of the AKT/mTOR signaling pathway, being a novel RNA oncogene.

Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of action. Herein we report that AKR1B10 activates lipid second messengers to stimulate cell proliferation. Our data showed that ectopic expression of AKR1B10 in breast cancer cells MCF-7 promoted lipogenesis and enhanced levels of lipid second messengers, including phosphatidylinositol bisphosphate (PIP2), diacylglycerol (DAG), and inositol triphosphate (IP3). In contrast, silencing of AKR1B10 in breast cancer cells BT-20 and colon cancer cells HCT-8 led to decrease of these lipid messengers. Qualitative analyses by liquid chromatography-mass spectrum (LC-MS) revealed that AKR1B10 regulated the cellular levels of total DAG and majority of subspecies. This in turn modulated the phosphorylation of protein kinase C (PKC) isoforms PKCδ (Thr505), PKCµ (Ser744/748), and PKCα/βII (Thr638/641) and activity of the PKC-mediated c-Raf/MEK/ERK signaling cascade. A pan inhibitor of PKC (Go6983) blocked ERK1/2 activation by AKR1B10. In these cells, phospho-p90RSK, phospho-MSK, and Cyclin D1 expression was increased by AKR1B10, and pharmacological inhibition of the ERK signaling cascade with MEK1/2 inhibitors U0126 and PD98059 eradicated induction of phospho-p90RSK, phospho-MSK, and Cyclin D1. In breast cancer cells, AKR1B10 promoted the clonogenic growth and proliferation of breast cancer cells in two-dimension (2D) and three-dimension (3D) cultures and tumor growth in immunodeficient female nude mice through activation of the PKC/ERK pathway. These data suggest that AKR1B10 stimulates breast cancer cell growth and proliferation through activation of DAG-mediated PKC/ERK signaling pathway.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function. Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression. Results: PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1MUT but not NOTCH1WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1WT HNSCC but had little effect as single agents. Conclusions: Our findings suggest that NOTCH1MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1WT HNSCC to PI3K/mTOR pathway inhibitors.

Aldo-keto reductase 1B10 (AKR1B10) is not expressed in normal breast, but upregulated in primary and metastatic breast cancers, being a negative prognostic factor. This study characterized the molecular mechanisms of AKR1B10-promoted breast cancer metastasis. Ectopic expression of AKR1B10 in breast cancer cells MCF-7 and MDA-MB-231 or siRNA-mediated silencing in BT-20 cells affected cell adhesion, migration and invasion in cell culture, and metastasis to the lung in the nude mice through upregulation of integrin α5 and δ-catenin. Silencing of integrin α5 or δ-catenin eradicated the cell adhesion and migration enhanced by AKR1B10, both of which acted synergistically. In these cells, the integrin α5 mediated focal adhesion kinase (FAK) signaling pathway was activated by AKR1B10, which, along with δ-catenin, stimulated Rac1-mediated cell migration and movement. In human primary and lymph node metastatic breast cancer, AKR1B10, integrin α5 and δ-catenin were correlatively upregulated with r=0.645 (p

Background Nasopharyngeal carcinoma (NPC) is one of the most common cancers in Southern China. Aldo-keto reductase 1B10 (AKR1B10) is upregulated in multiple tumors and plays an oncogenic role. Objective To examine the expression of AKR1B10 at mRNA and protein levels in nasopharyngeal tumors and correlate its expression with clinicopathological parameters. Methods A tissue microarray, paraffin blocks, and frozen surgical nasopharyngeal samples were procured. Western blot and immunohistochemistry were used to estimate AKR1B10 protein expression, and mRNA levels were detected by real time RT-PCR. Results We found that AKR1B10 expression was increased in malignant tissues compared to the normal tissues (p= 0.000). In NPC tissues, AKR1B10 expression appeared high specifically in squamous cell carcinoma, but low in basal cell carcinoma, adenoid cystic carcinoma, adenocarcinoma and undifferentiated carcinoma (p= 0.000). AKR1B10 expression also demonstrated correlation with tumor differentiation, with a high level in well and moderately differentiated but a low level in poorly differentiated carcinoma (p= 0.000). AKR1B10 was also upregulated in hyperplasia and benign tumors (p= 0.000), and demonstrated a specific nuclear distribution in these non-cancerous diseases. Conclusions AKR1B10 is overexpressed in nasopharyngeal hyperplasia, benign tumors, and carcinomas, being a potential new biomarker.

Hundreds of NF-κB inhibitors have been developed for cancer therapy, but their clinical efficacy is unsatisfactory. Here we show that the phosphorylation activation at Ser536 of RelA/p65 protein, a main subunit in the NF-κB family, may play a tumor-suppressive role. In normal colon mucosa, RelA/p65 phosphorylation at Ser536 was increasingly increased with the maturation and apoptotic shedding of epithelial cells, but the phosphorylation at Ser536 was decreased in colon cancer. In colon (HCT116 p53 wt and p53 -/-), breast (MCF7), and prostate (LNCaP and DU145) cancer cells, a phosphomimetic mutation of RelA/p65 at Ser536 (named p65/S536D) triggered dramatic apoptosis through affecting expression of a wide range of cell death/survival genes, such as Bim, Puma, Noxa, Bcl-2 and survivin. In HCT116 cells, p65/S536D mutant upregulated Fas, insulted mitochondrial membrane potential, and triggered cleavage and activation of caspase-3, 7, 8 and 9. A FasL neutralizing antibody (NOK1) prevented cell death induced by the p65/S536D. A pan inhibitor of caspases, Z-VAD-FMK (20 μM), blocked caspase-mediated mitochondrial membrane depolarization. This p65/S536D also triggered senescence in HCT116 cells through a p16-dependent pathway, but not in MFC7 due to lack of p16. Intratumoral delivery of the p65/S536D effectively suppressed tumor growth in nude mice. Together our data suggest that the phosphorylation of RelA/p65 at Ser536 may confer it a tumor-suppressive role by inducing apoptosis and senescence, highlighting the importance of discriminating the function and active status of individual active sites in RelA/p65 when NF-κB inhibitors are considered for targeted therapy of cancer.

Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs have been characterized to date. A novel lncRNA transcript, 2.53 kb in length, was identified by transcriptome sequencing analysis, and was named p53-inducible cancer-associated RNA transcript 1 (PICART1). PICART1 was found to be upregulated by p53 through a p53-binding site at -1808 to -1783 bp. In breast and colorectal cancer cells and tissues, PICART1 expression was found to be decreased. Ectopic expression of PICART1 suppressed the growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated cell growth and migration. In these cells, the expression of PICART1 suppressed levels of p-AKT (Thr308 and Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, while p21Waf/cip1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor-suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade.

Inactivation of p53 occurs frequently in various cancers. RITA is a promising anticancer small molecule that dissociates p53-MDM2 interaction, reactivates p53 and induces exclusive apoptosis in cancer cells, but acquired RITA resistance remains a major drawback. This study found that the site-differential phosphorylation of nuclear factor-κB (NF-κB) RelA/p65 creates a barcode for RITA chemosensitivity in cancer cells. In naive MCF7 and HCT116 cells where RITA triggered vast apoptosis, phosphorylation of RelA/p65 increased at Ser536, but decreased at Ser276 and Ser468; oppositely, in RITA-resistant cells, RelA/p65 phosphorylation decreased at Ser536, but increased at Ser276 and Ser468. A phosphomimetic mutation at Ser536 (p65/S536D) or silencing of endogenous RelA/p65 resensitized the RITA-resistant cells to RITA while the phosphomimetic mutant at Ser276 (p65/S276D) led to RITA resistance of naive cells. In mouse xenografts, intratumoral delivery of the phosphomimetic p65/S536D mutant increased the antitumor activity of RITA. Furthermore, in the RITA-resistant cells ATP-binding cassette transporter ABCC6 was upregulated, and silencing of ABCC6 expression in these cells restored RITA sensitivity. In the naive cells, ABCC6 delivery led to RITA resistance and blockage of p65/S536D mutant-induced RITA sensitivity. Taken together, these data suggest that the site-differential phosphorylation of RelA/p65 modulates RITA sensitivity in cancer cells, which may provide an avenue to manipulate RITA resistance.

Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at V(max) of 837.42±81.39nmol/mg/min, K(m) of 9.317±2.25mM and k(cat)/K(m) of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with V(max) at 460.23±28.12nmol/mg/min, K(m) at 0.461±0.09mM and k(cat)/K(m) at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50nM) and idarubicin (30nM) to corresponding alcohols. Within 24h, approximately 20±2.7% of daunorubicin (1μM) or 23±2.3% of idarubicin (1μM) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7±0.9% and 5±1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggest that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin.

Over the past decade, The Cancer Genome Atlas (TCGA) has profiled more than 11,000 tumors spanning 33 distinct cancer types. The TCGA PanCanAtlas is a collaborative project by the TCGA Research Network that aims to address relevant overarching questions in oncology based on a cross-cancer analysis of the full, uniformly reprocessed TCGA data set. Here, we present results from our analysis of genetic alterations in mitogenic signaling pathways across cancer. Genetic alterations in signaling pathways that control cell cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations and copy-number changes in 9,125 tumor samples profiled by TCGA, we analyzed the mechanisms and patterns of alterations in 10 canonical pathways: cell cycle, Hippo, Myc, Notch, beta-catenin / WNT, PI-3-Kinase / Akt, receptor-tyrosine kinase / RAS / MAP-kinase signaling, TP53, and TGF-beta signaling, as well as oxidative stress response. For each of these pathways, we propose an expert-curated description (or “template”) that includes the relevant (altered) genes and the connections between them, as well as a detailed catalogue of the driver mutations and copy number changes with known oncogenic relevance. We provide a high-level map of pathway alteration frequencies across tissues and relevant cancer subtypes as well as detailed frequencies of alteration at the gene level for each individual pathway. We also investigate relationships of co-occurrence and mutual exclusivity across pathways and evaluate therapeutic implications, including drug combinations. Forty-nine percent of tumors had at least one potentially targetable alteration in the evaluated pathways, and 31% of tumors had multiple targetable alterations, making them candidates for combination therapy. Our work delineates the full landscape of oncogenic alterations in mitogenic signaling pathways across cancer, and the pathway templates as well as the richly annotated data set that we provide will constitute an invaluable public resource for future use by the cancer genomics and precision oncology communities. Citation Format: Francisco Sanchez-Vega, Marco Mina, Joshua Armenia, Walid K. Chatila, Augustin Luna, Konnor La, Sofia Dimitriadoy, David L. Liu, Havish S. Kantheti, Zachary Heins, Angelica Ochoa, Benjamin Gross, Jianjiong Gao, Hongxin Zhang, Ritika Kundra, Cyriac Kandoth, Istemi Bahceci, Leonard Dervishi, Ugur Dogrusoz, Wanding Zhou, Hui Shen, Peter W. Laird, Alice H. Berger, Trever G. Bivona, Alexander J. Lazar, Gary Hammer, Thomas Giordano, Lawrence Kwong, Grant McArthur, Chenfei Huang, Mitchell J. Frederick, Frank McCormick, Matthew Meyerson, The Cancer Genome Atlas Research Network, Eliezer Van Allen, Andrew D. Cherniack, Giovanni Ciriello, Chris Sander, Nikolaus Schultz. The molecular landscape of oncogenic signaling pathways in The Cancer Genome Atlas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3302.

Background: The NOTCH1 gene functions as either an oncogene or tumor suppressor in cancer depending upon the tumor type. Our group previously characterized the genomic alterations in head and neck squamous cell carcinoma (HNSCC), discovering that NOTCH1 is frequently altered with a pattern of inactivating mutations suggesting it is a tumor suppressor in this cancer type. However, recent work by others suggests NOTCH1 signaling plays a more complex role, possibly promoting a more aggressive phenotype or cancer stem cell-like properties in HNSCCs with wild type NOTCH1. Our present study aimed to systematically compare the phenotypic consequences of NOTCH1 signaling in HNSCC to better understand its function in cancer, and identify targets downstream of NOTCH1 signaling. Methods: Established HNSCC cell lines wild type for NOTCH1 (PJ34, FADU) or harboring an inactivating mutation (UMSCC22A) were engineered to express activated cleaved NOTCH1 (cl-NOTCH1) from a doxycycline-inducible promoter. In vitro cell growth was measured with clonogenic assays. Stem cell-like properties were measured by orosphere formation and anoikis resistance. Stem cell markers for HNSCC including Aldehyde dehydrogenase activity (ALDH), CD133, and CD44 expression were measured by flow cytometry. NOTCH1-regulated downstream gene expression changes were examined by RNA-seq and qRT-PCR. Results: Activation of NOTCH1 inhibited clonogenic growth of all three cell lines, regardless of original NOTCH1 gene status. Growth inhibition was frequently accompanied by spontaneous formation of spheroid-like structures, characteristic of stem cells. NOTCH1 activation in UMSCC22A and FADU cells promoted orosphere formation and anoikis resistance, conveying some stem cell-like properties. However, classical stem cell markers including ALDH activity, CD133, and CD44 expression were not affected by NOTCH1 activation. Furthermore, RNA-seq demonstrated that critical cancer-associated pathways, including proliferation, differentiation, and migration, were regulated by NOTCH1. NOTCH1 activation downregulated gene expression of ITGA3, ITGA4, ITGB1, ITGB6, and LAMC2, which are key adhesion molecules that human basal keratinocytes use for attachment to the basement membrane and maintenance of the stem cell compartment. Concomitantly, NOTCH1 activation increased the basal/superbasal marker SOX2, but also the early differentiation markers KRT4 and KRT13. SiRNA-mediated SOX2 silencing blocked NOTCH1-promoted anoikis resistance. Conclusion: NOTCH1 activation inhibits in vitro growth regardless of mutational status. We hypothesize that stem cell-like properties associated with NOTCH1 activation in HNSCC may be a consequence of pathways that recapitulate early differentiation, rather than true stem cell maintenance. Citation Format: Chenfei Huang, Shhyam Moorthy, Qiuli Li, Rami Saade, Jiping Wang, Xiayu Rao, Noriaki Tanaka, Jiexin Zhang, Lin Tang, Curtis R. Pickering, Patrick A. Zweidler-McKay, Abdullah A. Osman, Tong-Xin Xie, Eve Shinbrot, Liu Xi, David Wheeler, Adel K. El-Naggar, Jing Wang, Jeffrey N. Myers, Mitchell J. Frederick. NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5506.

Purpose: Ulcerative colitis and colitis-associated colorectal cancer (CAC) is a serious health issue, but etiopathological factors remain unclear. Aldo-keto reductase 1B10 (AKR1B10) is specifically expressed in the colonic epithelium, but downregulated in colorectal cancer. This study was aimed to investigate the etiopathogenic role of AKR1B10 in ulcerative colitis and CAC. Experimental Design: Ulcerative colitis and CAC biopsies (paraffin-embedded sections) and frozen tissues were collected to examine AKR1B10 expression. Aldo-keto reductase 1B8 (the ortholog of human AKR1B10) knockout (AKR1B8−/−) mice were produced to estimate its role in the susceptibility and severity of chronic colitis and associated dysplastic lesions, induced by dextran sulfate sodium (DSS) at a low dose (2%). Genome-wide exome sequencing was used to profile DNA damage in DSS-induced colitis and tumors. Results: AKR1B10 expression was markedly diminished in over 90% of ulcerative colitis and CAC tissues. AKR1B8 deficiency led to reduced lipid synthesis from butyrate and diminished proliferation of colonic epithelial cells. The DSS-treated AKR1B8−/− mice demonstrated impaired injury repair of colonic epithelium and more severe bleeding, inflammation, and ulceration. These AKR1B8−/− mice had more severe oxidative stress and DNA damage, and dysplasias were more frequent and at a higher grade in the AKR1B8−/− mice than in wild-type mice. Palpable masses were seen in the AKR1B8−/− mice only, not in wild-type. Conclusions: AKR1B8 is a critical protein in the proliferation and injury repair of the colonic epithelium and in the pathogenesis of ulcerative colitis and CAC, being a new etiopathogenic factor of these diseases. Clin Cancer Res; 21(6); 1466–76. ©2014 AACR.

Hundreds of NF-κB inhibitors have been developed for cancer therapy, but their clinical efficacy is unsatisfactory. Here we show that the phosphorylation activation at Ser536 of RelA/p65 protein, a main subunit in the NF-κB family, may play a tumor-suppressive role. In normal colon mucosa, RelA/p65 phosphorylation at Ser536 was increasingly increased with the maturation and apoptotic shedding of epithelial cells, but the phosphorylation at Ser536 was decreased in colon cancer. In colon (HCT116 p53 wt and p53 -/-), breast (MCF7), and prostate (LNCaP and DU145) cancer cells, a phosphomimetic mutation of RelA/p65 at Ser536 (named p65/S536D) triggered dramatic apoptosis through affecting expression of a wide range of cell death/survival genes, such as Bim, Puma, Noxa, Bcl-2 and survivin. In HCT116 cells, p65/S536D mutant upregulated Fas, insulted mitochondrial membrane potential, and triggered cleavage and activation of caspase-3, 7, 8 and 9. A FasL neutralizing antibody (NOK1) prevented cell death induced by the p65/S536D. A pan inhibitor of caspases, Z-VAD-FMK (20 μM), blocked caspase-mediated mitochondrial membrane depolarization. This p65/S536D also triggered senescence in HCT116 cells through a p16-dependent pathway, but not in MFC7 due to lack of p16. Intratumoral delivery of the p65/S536D effectively suppressed tumor growth in nude mice. Together our data suggest that the phosphorylation of RelA/p65 at Ser536 may confer it a tumor-suppressive role by inducing apoptosis and senescence, highlighting the importance of discriminating the function and active status of individual active sites in RelA/p65 when NF-κB inhibitors are considered for targeted therapy of cancer.

Pancreatic cancer (PC) is a fatal disease with a high mortality and poor prognosis. PC is the fourth leading cause of cancer death in America, and 80 % of PCs are diagnosed at an unresectable stage. Effective early detection assays are crucial since a successful operation at early stage is the best strategy for this disease. S100 calcium-binding protein P (S100P) has been reported as a predictive diagnostic index for PC and involves in the development of PC. However, the diagnostic accuracy of S100P in detecting PC has never been systematically assessed. The aim of the present study was to evaluate the diagnostic performance of S100P for PC. All relevant original articles about S100P in the diagnosis of PC published up to December 2013 were retrieved. The methodological quality of each study was assessed by QUADAS. The overall diagnostic sensitivity, specificity, diagnostic odds ratio (DOR), with 95 % confidence interval (CI), and area under the receiver operating characteristic curve (AUC) were pooled to evaluate the diagnostic value of S100P for PC using the Meta-DiSc1.4 statistical software. Eight studies met our criteria in the present meta-analysis. The pooled sensitivity, specificity, and DOR calculated by the bivariate random effects model were 0.87 (95 % CI 0.83-0.90), 0.88 (95 % CI 0.82-0.93), and 38.32 (95 % CI 11.22-130.87), respectively. The summary receiver operating characteristic (SROC) curve was located near the desirable left corner and the AUC was 0.9272. The current evidence suggests that S100P plays an important role in the diagnosis of PC with a high sensitivity and specificity. S100P may be regarded as a promising diagnostic marker to PC screening.

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50 ng/ml) and insulin (10 nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp −222 to −212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.

Aldo-keto reductase 1B10 (AKR1B10) is a secretory protein that is upregulated with tumorigenic transformation of human mammary epithelial cells. This study demonstrated that AKR1B10 was overexpressed in 20 (71.4%) of 28 ductal carcinomas in situ, 184 (83.6%) of 220 infiltrating carcinomas and 28 (87.5%) of 32 recurrent tumors. AKR1B10 expression in breast cancer was correlated positively with tumor size (p = 0.0012) and lymph node metastasis (p = 0.0123) but inversely with disease-related survival (p = 0.0120). Univariate (p = 0.0077) and multivariate (p = 0.0192) analyses both suggested that AKR1B10, alone or together with tumor size and node status, is a significant prognostic factor for breast cancer. Silencing of AKR1B10 in BT-20 human breast cancer cells inhibited cell growth in culture and tumorigenesis in female nude mice. Importantly, AKR1B10 in the serum of breast cancer patients was significantly increased to 15.18 ± 9.08 ng/ml [n = 50; 95% confidence interval (CI), 12.60-17.76], with a high level up to 58.4 ng/ml, compared to 3.34 ± 2.27 ng/ml in healthy donors (n = 60; 95% CI, 2.78-3.90). In these patients, AKR1B10 levels in serum were correlated with its expression in tumors (r = 0.8066; p < 0.0001). Together our data suggests that AKR1B10 is overexpressed in breast cancer and may be a novel prognostic factor and serum marker for this deadly disease.

Background Constitutive photomorphogenesis protein 1 (COP1; also known as Rfwd2), an evolutionally conserved E3 ubiquitin ligase, has been defined to mediate the degradation of several important proteins, such as p53, c-Jun, ETS, FOXO1 and ACC1, being involved in tumorigenesis. In response to DNA damage, COP1 is autodegraded in the context of ATM, which is triggered by 14-3-3σ. However, the biological function and expression regulation of COP1 remain to be fully understood. This study reports a novel function of COP1 in lipid metabolism. Methods We targeted COP1 expression in cancer cells MCF-7 and HepG2 with COP1 shRNA lentivirus and COP1 overexpression lentivirus, respectively. We investigated the lipid synthesis, glucose oxidation and conversion into lipids in these cells using radiolabeled acetate and glucose and lipid complex analysis. Quantitative real-time PCR and Western blot were used to examine gene expression. Acetyl CoA carboxylase-1 (ACC1) siRNA and TOFA, an inhibitor of ACC1, were used to modulate the intracellular lipid synthesis. Results Lipid depletion induced by acetyl CoA carboxylase-1 (ACC1) knockdown or the inhibition of ACC1 by TOFA induced COP1 expression, but palmatic acid, the end-product of de novo fatty acid synthesis pathway, reversed TOFA-induced COP1 expression. In addition, U73122, an inhibitor of phospholipase C that cleaves PIP2 into DAG and IP3, triggered COP1 expression. These data suggest that COP1 expression is mediated by cellular lipid state, and the lipid second messengers (PIP2 and DAG) are involved in the expression regulation of COP1. In contrast, the ectopic expression of COP1 greatly reduced pAMPK and pACC1 levels, but elevated fatty acid synthase (FASN) expression, leading to the increase of fatty acid (FA)/lipid synthesis. In sharp contrast, siRNA-mediated COP1 knockdown in cancer cells significantly increased pACC1 and pAMPK levels and reduced FASN expression, leading to decrease of lipid synthesis. The cell growth and proliferation was accordingly changed in response to the alterations of lipid synthesis. Altogether our data suggest that COP1 expression is regulated through the lipid second messenger mediated signaling, and the upregulated COP1 in turn enhances lipid synthesis, functioning as a positive feedback. Conclusion COP1 functions as a sensor of cellular lipid state and promoter of lipogenesis, facilitating tumorigenesis. Note: This abstract was not presented at the meeting. Citation Format: Chenfei Huang, Yiwen Bu, Deliang Cao. COP1 functions as a sensor of cellular lipids and regulator of lipogenesis in human cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1142. doi:10.1158/1538-7445.AM2015-1142

Bidirectional crosstalk between the estrogen receptor (ER) and HER2 has been demonstrated in preclinical model systems. We have previously shown in HER2+ breast cancer cells with low ER that potent HER2- targeted therapy results in ER reactivation that functions as an escape pathway to evade therapeutic inhibition. Here we investigated the role of HER pathway components in resistance to endocrine therapies using UACC812 cells, which are endogenously amplified for HER2. Endocrine resistant (R) derivatives of the UACC812 line were developed through long-term (>6 months) culturing with estrogen deprivation (ED) alone or with 4-OH-tamoxifen (Tam, 10-7M). Endocrine treatment [continued ED, estrogen (E2, 10-9M), Tam (10-7M), or fulvestrant (Ful, 10-7M)] of parental cells slowed cell growth compared to estrogen treatment (P200 days) complete regression in all mice. Our results further illustrate the plasticity of the ER and HER2 pathways and provide additional support for the role of the HER network, especially EGFR, in resistance to endocrine therapy in ER+/HER2+ breast cancer. The data emphasize the need for complete HER blockade for optimal therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-02-20.

About 25% of human breast cancers are amplified for HER2 with half of these tumors also expressing estrogen receptor (ER). Therapies targeting HER2 are very effective in the metastatic and the adjuvant settings, especially, although de novo or acquired resistance are still major problems. Trastuzumab (T) and lapatinib (L) are approved drugs now used in the clinic for treatment of HER2+ tumors. Data suggest that T works primarily by blocking signals generated by HER2 homodimers, while L is a small molecule tyrosine kinase inhibitor that more completely blocks the pathway by inhibiting HER1 in addition to HER2. In the clinic, these drugs demonstrate incomplete cross-resistance since L is active in some patients with T-resistant tumors. However, the mechanisms for this resistance have not been clarified.To investigate the mechanisms for acquired resistance, we developed a panel of HER2+ cell lines resistant to T, L, and L+T by long-term exposure to increasing drug concentration in vitro. Two of these lines, BT474 and UACC812, are amplified for HER2 and also express ER, and they, together with subclones resistant to L, T, and L+T, were used to better understand potential resistance mechanisms. Western blot analysis of the parental BT474 and its resistant subclones showed that subclones resistant to T had reactivated the HER2 signaling pathway, while subclones resistant to L or L+T in which the HER receptor layer was more completely inhibited showed continued complete blockade of the HER2 pathway at the receptor layer but high levels of ER activity and phosphorylated-AKT. L, but not L+T, subclones after more prolonged time in culture did reactivate the HER pathway. UACC812 resistant cells were similar to BT474: T-resistant clones showed evidence of reactivation of HER signaling while L and L+T resistant clones showed enhanced ER activity. These cells showed no reactivation of HER signaling even after prolonged exposure in vitro. Consistent with these data, both BT474 and UACC812 T-resistant clones were still sensitive to and cell proliferation was inhibited by L. L-resistant clones, however, were also resistant to T. The potent anti-estrogen fulvestrant (F) was used to evaluate the role of ER in these resistant clones. T-resistant clones from both parental lines were resistant to F, indicating that ER had no role in resistance. In contrast, L and L+T-resistant clones, but not parental cells, were extremely sensitive to F with significant inhibition of cell proliferation in vitro.These data demonstrate that only partial inhibition of the HER2 pathway in breast cancer cells by T can be overcome by activating other components of the HER pathway. Resistance to more complete HER2 blockade with L or L+T requires reactivation of a redundant cell survival pathway, in this case ER, which is upregulated by HER2 blockade. Optimal therapy in those tumors may require both ER and HER2-targeted therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 708.