bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1745: Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells
Authors: Frock Richard L. , Kumar Vipul , Liang Zhouyi , Zurita Jeff , Du Zhou , Lin Sherry G. , Boboila Cristian , Alt Frederick W.
Published: August 2019

Classical non-homologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but is thought to predominate in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase4 core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG-initiated DSBs during V(D)J recombination in G1-phase lymphocyte progenitors, cycling cells deficient for core C-NHEJ factors join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination to C-NHEJ has been attributed to RAG-mediated exclusion of A-EJ; however, it remains unclear whether A-EJ is similarly excluded from more general DSBs in G1. Here, we report that Ku actively and robustly suppresses A-EJ of both RAG- and Cas9-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while Cas9/gRNA DSBs remain as free broken ends in Ligase4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Ligase4-deficient cells restores DSB re-joining and translocation to levels observed in Ku70-deficient counterparts. Correspondingly, while V(D)J recombination is abrogated in Ligase4-deficient lines, V(D)J-like joining occurs in Ku70-deficient and Ku70/Ligase4-double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Ligase4-deficient progenitor B cells are functionally end-joining deficient due to a complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase4 deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including ability of Ku-deficiency to rescue Ligase4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase.

Citation Format: Richard L. Frock, Vipul Kumar, Zhouyi Liang, Jeff Zurita, Zhou Du, Sherry G. Lin, Cristian Boboila, Frederick W. Alt. Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1745.
Abstract 1748: RAD51 has an anti-recombinase activity that is countered by BRCA2 - In vitro
Authors: Wang Charles , Jimenez-Sainz Judit , Jensen Ryan B. , Mazin Alexander A.
Published: August 2019

Homologous Recombination (HR) is a high-fidelity process with a range of biologic functions from generation of genetic diversity to repair of DNA double-strand breaks (DSBs). In mammalian cells, BRCA2 facilitates the polymerization of RAD51 onto ssDNA to form a presynaptic nucleoprotein filament. This filament can then strand invade a homologous dsDNA to form the displacement loop (D-loop) structure leading to the eventual DSB repair. This pro-recombinatory feature of RAD51-BRCA2 is well documented. Here, we present biochemical evidence that RAD51 can cause D-loop disassembly. Furthermore, we show that this RAD51 anti-recombinatory activity is countered by BRCA2. These results demonstrate that BRCA2 may have a previously unexpected role: regulation of HR at a post-synaptic stage by modulating RAD51-mediated D-loop dissociation. Our results provide a biochemical mechanistic understanding of homeostasis between RAD51 and BRCA2, which has been suggested to be an important factor of HR based on previously published cellular data.

Citation Format: Charles Wang, Judit Jimenez-Sainz, Ryan B. Jensen, Alexander A. Mazin. RAD51 has an anti-recombinase activity that is countered by BRCA2 - In vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1748.
Abstract 1750: USP22 directly interacts with PALB2 and is required for efficient homologous recombination of DNA double-strand breaks
Authors: Nardi Isaac K. , Stark Jeremy , Larsen Adrien , Raz Dan
Published: August 2019

DNA damage by environmental stressors and agents is a huge problem for cells and must be repaired quickly for the cell to maintain its integrity. Cells employ several repair pathways and mechanisms to rectify damaged DNA. Homologous Recombination (HR) repair comprises a series of pathways in a cell that can function to repair DNA double-stranded breaks (DSBs) through template-dependent mechanisms to preserve genomic integrity. Failure to repair DSBs can result in chromosomal loss, carcinogenesis, and apoptosis. Deubiquitylases (DUBs) are a family of proteases that cleave ubiquitin from proteins and have been implicated in several biological processes including tumor development, cell growth, cell cycle regulation, and signal transduction. There is a growing catalog of studies showing the vital role of DUB’s in the DNA Damage Response (DDR). But, given that there are over 100 DUBs in the human genome their functions in DDR have still not been fully fleshed out. Along with its role in antibody class switch repair, USP22 is a DUB that has been implicated as a putative oncogene with over-expression observed in various cancer types and is associated with a poor patient prognosis. Furthermore, over-expression of USP22 leads to cisplatin resistance in lung cancer cells strongly suggesting it plays in a vital role in DDR and chemoresistance in recurrent cancers. In this study we show USP22 is necessary for HR, localizes to sites of DNA damage, and is necessary for recruitment of the PALB2-BRCA2-Rad51 complex through stabilizing PALB2 protein levels. Lastly, we show USP22 directly binds to the WD40 domain of PALB2 and this binding stimulates USP22 catalytic activity. Taken together, these findings show a new and novel role of USP22 in the HR pathway.

Note: This abstract was not presented at the meeting.

Citation Format: Isaac K. Nardi, Jeremy Stark, Adrien Larsen, Dan Raz. USP22 directly interacts with PALB2 and is required for efficient homologous recombination of DNA double-strand breaks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1750.
Abstract 1760: Plasma vitamin D levels and DNA repair capacity in four molecular subtypes of women with breast cancer
Authors: Ortiz Carmen , Encarnacion Jarline , Vergne Ralphdy , Padilla Luis , Matta Jaime
Published: August 2019

Background: Vitamin D regulates estrogen synthesis among other mechanisms involved in breast cancer (BC) carcinogenesis. An important gap, to our knowledge, is whether blood levels of vitamin D are associated with DNA repair capacity in women with BC. Previously, we reported that a low DNA repair capacity (DRC) is a risk factor for BC and that DRC levels are associated with estrogen receptor (ER) positivity in women with BC. Moreover, we also reported that there is an intrinsic variability in DRC levels among the four principal BC subtypes. Therefore, our aim is to study the link between DRC and plasma vitamin D levels in BC and among BC subtypes.

Methods: Treatment-naïve BC cases and controls were selected from our cohort of Hispanic women (n=1,187). DRC was assessed in lymphocytes through the host-cell reactivation assay. Vitamin D was measured using LC/MS/MS.

Results: Women with BC (n=91) had higher (+6.6 ng/ml) vitamin D than controls (n=22) (p=0.007). There were significant differences in vitamin D levels (p<0.05) between BC cases with high (≥3.8%) (n=17) and low (<3.8%) (n=74) DRC levels. Vitamin D and DRC were negatively correlated in BC cases (r=-0.2296, p=0.03). When women with BC and low DRC levels (n=56) were stratified into: luminal A (n=17), luminal B (n=11), HER2+ (n=10), and triple-negative (n=18), women with HER2+ and TNBC showed the highest levels of vitamin D (p<0.05). Women with ER- had higher levels (+7.6 ng/ml) of vitamin D than women with ER+ tumors (p=0.005). After finding that plasma vitamin D levels from women with BC and controls were significantly different when stratified by DRC levels and BC subtypes, ROC curves were constructed to examine whether these levels could allow us to discriminate between our four study groups. Significant differences (p=0.0126) were found for cases and controls with low DRC; AUC of 0.7178. When considering all women with BC independently of DRC levels, the AUC was 0.7353 (p=0.0300).

Conclusions: This study provides the first evidence of a link between vitamin D and DRC levels in women with BC. We also present data as to how vitamin D varies across four molecular subtypes of BC in Hispanic women.

Impact: Our results fills an important gap on the link between plasma vitamin D and DRC levels in women with BC.

Funding: This study was supported by grants #S06GM008239-20, 9SC1CA182846-04, and 5SC1CA157250-02, 5U54CA163071-04, 5R25GM096955-08.

Citation Format: Carmen Ortiz, Jarline Encarnacion, Ralphdy Vergne, Luis Padilla, Jaime Matta. Plasma vitamin D levels and DNA repair capacity in four molecular subtypes of women with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1760.
Abstract 1762: The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1
Authors: Sanchez-Solana Beatriz , Wang Dunrui , Qian Xiaolan , Papageorge Alex , Velayoudame Parthibane , Acharya Jairaj , Lowy Douglas R.
Published: August 2019

The tumor suppressor gene DLC1 encodes a multi-domain protein, which includes a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, B, and C, and has been hypothesized to be the basis of its tumor suppressor functions. DLC1 also contains a less well characterized START (StAR-related lipid transfer) domain in its C-terminus, whose overall contribution to DLC1 function remains incompletely understood. START domains in other proteins have been shown to bind lipids, but no lipid has yet been identified that binds the DLC1 START domain. In the present study, we attempted to gain further insight into the molecular function and lipid binding properties of the DLC1 START domain.

We previously determined that Caveolin-1, the main structural component and marker of caveolae, interacts with the DLC1 START domain, and this interaction contributes to the full tumor suppressor activity of DLC1. In unpublished studies, we additionally identified Phospholipase C delta 1 (PLCD1), which participates in intracellular Ca2+ mobilization, forming a complex with the DLC1 START domain and with Caveolin-1.

We have now developed evidence that, in addition to binding Caveolin-1 and PLCD1, the DLC1 START domain binds a lipid, phosphatidylserine (PS), and have identified a colon cancer-associated DLC1-START domain mutant (R947C) that results in a protein deficient in forming a complex with all three binding partners and displays reduced tumor suppressor activity but intact RhoGAP activity. The interaction between the DLC1-START domain and PS can occur independently of PLCD1 and/or Caveolin-1, but PS promotes complex formation between the DLC1 START domain and PLCD1 or Caveolin-1, without altering PLCD1 to Caveolin-1 binding. Biological assays with wild type DLC1 show cooperation between DLC1 and PLCD1 or Caveolin-1 to inhibit cell migration, but no cooperation is observed when all three genes are overexpressed, suggesting that Caveolin-1 and PLCD1 occupy the same migration signaling pathway. Thus, we have identified a biologically relevant complex between DLC1 and three macromolecules (PLCD1, PS and Caveolin-1) that contributes to the full tumor suppressor function of DLC1 independently of its RhoGAP activity, and are testing the hypothesis that PS serves as a linker between the DLC1 START domain and PLCD1 or Caveolin-1.

Citation Format: Beatriz Sanchez-Solana, Dunrui Wang, Xiaolan Qian, Alex Papageorge, Parthibane Velayoudame, Jairaj Acharya, Douglas R. Lowy. The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1762.
Abstract 1764: Targeting nucleotide exchange to inhibit Gq/11 driver mutations in uveal melanoma
Authors: Onken Michael D. , Makepeace Carol M. , Kaltenbronn Kevin M. , Kanai Stanley M. , Todd Tyson D. , Wang Shiqi , Broekelmann Thomas J. , Rao Prabakar Kumar , Cooper John A. , Blumer Kendall J.
Published: August 2019

Constitutively activating mutations in the G-protein alpha subunits GNAQ(Gαq) and GNA11(Gα11) act as oncogenic drivers in over 90% of uveal (eye) melanoma (UM) tumors. We show that constitutively active Gαq and Gα11 can be targeted in UM cells by the cyclic depsipeptide FR900359 (FR). FR inhibits GDP/GTP guanine nucleotide exchange allosterically to trap constitutively active Gαq/11 in inactive GDP-bound Gαβγ heterotrimers. FR inhibits second messenger signaling, arrests proliferation and reinstates melanocytic differentiation in UM cells driven by constitutively active Gαq or Gα11. At higher doses, FR also induces apoptosis. The re-differentiation and anti-proliferative effects of FR are not seen in UM cells that lack mutations in Gαq or Gα11. FR promotes UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)-mediated gene silencing, and this re-differentiation can be blocked with an EZH2 inhibitor. The effector system regulating PRC2 downstream of constitutively active Gαq/11 in UM is currently under investigation. Preliminary data from human primary tumor samples suggest that targeting constitutively active Gαq/11 with FR could provide an important therapeutic approach for UM.

Citation Format: Michael D. Onken, Carol M. Makepeace, Kevin M. Kaltenbronn, Stanley M. Kanai, Tyson D. Todd, Shiqi Wang, Thomas J. Broekelmann, Prabakar Kumar Rao, John A. Cooper, Kendall J. Blumer. Targeting nucleotide exchange to inhibit Gq/11 driver mutations in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1764.
Abstract 177: Sulfiredoxin promotes the invasion of human colorectal cancer cells through the activation of fascin
Authors: Jiang Hong , Ying Qi , Evers B. Mark , Wei Qiou
Published: August 2019

Sulfiredoxin (Srx) is multifunction enzyme with a primary antioxidant role of reducing the overoxidized inactive form of peroxiredoxins (Prxs). In contrast to the well-studied biochemical function, the biological significance of Srx in human diseases including cancer has not been well studied. Our previous studies show that it plays an important role in tumorigenesis and malignant progression of human skin, lung and colorectal cancers. Fascin homologue 1 (FSCN1) is an actin-binding protein that is critical for cancer cell invasion and metastasis. The purpose of this study is to understand the molecular mechanism of Srx on the invasion and metastasis of human colorectal cancer cells through the regulation of FSCN1. The effects of Srx on the migration and invasion of human colon cancer HCT116 cells were evaluated by wound healing and matrigel invasion assay, respectively. Actin stress fiber was visualized by F-actin fluorescence staining. Western blotting and reverse transcription & real-time PCR were used to examine the protein and RNA levels of FSCN1 in HCT116 cells. The effect of Srx on FSCN1 promoter and 3’ UTR were determined using luciferase reporter construct. We demonstrated that (1) knockdown of Srx suppressed HCT116 colony formation and cell invasion in the matrigel invasion assay; (2) knockdown of Srx inhibited FSCN1 expression in HCT116 cells at both RNA and protein level; (3) the inhibition is the result of decreased FSCN1 mRNA stability; (5) Srx stabilizes FSCN1 mRNA 3’ UTR mediated reporter expression; (4) knockdown of Srx reduced actin stress-fiber formation; (5) Srx and Prx4 synchronically regulates FASN1 expression. Therefore, we concluded that Srx promotes the invasion and metastasis of human colon cancer cells through the upregulation of FSCN1.

Citation Format: Hong Jiang, Qi Ying, B. Mark Evers, Qiou Wei. Sulfiredoxin promotes the invasion of human colorectal cancer cells through the activation of fascin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 177.
Abstract 1770: RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation
Authors: Yao Ke , Lee Sung-Young , Peng Cong , Lim Do Young , Yamamoto Hiroyuki , Ryu Joohyun , Lim Tae-Gyu , Chen Hanyong , Ma Wei-Ya , Bode Ann M. , Dong Zigang
Published: August 2019

Inflammation is a complex biological host reaction to tissue damage, infection and trauma. Extensive study of the inflammatory response has led to the identification of several protein kinases that are essential for signaling and could be potential therapeutic targets. The RSK family of kinases has multiple cellular functions. In our study, we found that RSK2 is a mediator for inflammation signaling and interacts with TRAF6. In vitro kinase assay results indicated that RSK2 strongly phosphorylates TRAF6 at serines 46, 47 and 48. Ectopic over-expression of TRAF6 or knocking down RSK2 expression confirmed that RSK2 is a positive regulator of TRAF6 K63 ubiquitination. TRAF6 is also required for RSK2 ubiquitination. TRAF6 bridges the TNF receptor superfamily and intracellular signaling for the induction of pro-inflammatory cytokines. We developed a colon inflammation model using RSK2 wild type (WT) and knockout (KO) mice. As expected, F4/80 and CD3 infiltration were significantly upregulated in WT mice compared to RSK2 KO mice. Furthermore, inflammation signaling, including Ikkα/β, p38 and JNKs, was dramatically up-regulated in WT mice. Colon tissue immunoprecipitation results further confirmed that TRAF6 K63 ubiquitination was lower in RSK2 KO mice. Overall, these results indicate that phosphorylation of TRAF6 (S46, 47, 48) by RSK2 is required for TRAF6 K63 ubiquitination and inflammation signaling.

Citation Format: Ke Yao, Sung-Young Lee, Cong Peng, Do Young Lim, Hiroyuki Yamamoto, Joohyun Ryu, Tae-Gyu Lim, Hanyong Chen, Wei-Ya Ma, Ann M. Bode, Zigang Dong. RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1770.
Abstract 1776: An investigation of the effects of the atypical protein kinase C-ζinhibitor ζ-Stat on clear cell carcinoma ovarian cancer proliferation and invasion
Authors: Smalley Tracess , Patel Rekha , Acevedo-Duncan Mildred
Published: August 2019

Ovarian cancer is the most lethal gynecological cancer diagnosis. There is no efficient screening process for ovarian cancers and the average stage of diagnosis is stage III. The most common ovarian cancer diagnosis is epithelial ovarian carcinomas, which constitute for 85-90% of prognosis; of this percent, clear cell ovarian carcinoma (CCOC) represents 5% of incidence. CCOC presents unique pathological features, has poor prognosis and has a high reoccurrence rate after treatment. An overexpression of atypical protein kinase C isoforms (PKC-ζ and PKC-ι/λ) has been observed in various malignant cells lines and are linked to pathways for cellular proliferation and invasion. In this investigation, ovarian cell lines (TOV21G and ES-2) were treated with the atypical PKC-ζ inhibitor ζ-Stat and assayed to determine the effects on proliferation and cellular invasion. These assays included cell counting, WST-1 assay, Western Blot and endpoint polymerase chain reaction. Mouse xenograft experiments were also performed to determine the effects of ζ-Stat on TOV21G xenograft tumor growth in vivo. Our data showed that ζ-Stat decreased the proliferation and cell viability of clear cell carcinoma ovarian cancer cells. In addition, it knocked down the protein expression of PKC-ζ, as well as the protein and mRNA levels of RhoA (Ras homolog Family Member A). Our results also showed that tumor growth in athymic female mice was decreased when treated with ζ-Stat and that mouse body weight was maintained. This suggests that PKC-ζ is a novel target in the carcinogenesis of CCOC and its inhibition by way of ζ-Stat decreased the rate of proliferation, tumor growth and expression of invasive protein pathways.

Citation Format: Tracess Smalley, Rekha Patel, Mildred Acevedo-Duncan. An investigation of the effects of the atypical protein kinase C-ζinhibitor ζ-Stat on clear cell carcinoma ovarian cancer proliferation and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1776.
Abstract 1777: Correlation between different C-KIT exon mutation and clinical outcome to Imatinib Mesylate treatment in patients with gastro-intestinal stromal tumors (GIST)
Authors: Bahnnasy Abeer A. , Khorshid Ola , Abdellateif Mona S. , Zakaria Ghada Y. , Allahloubi Nasr M. , Nassar Auhood , Zekri Abdel-Rahman N.
Published: August 2019

Aim: C-KIT exon mutations were assessed in patients with gastrointestinal stromal tumor (GIST) in relation to patients’ characteristics, response to treatment and survival rates.

Methods: C-KIT mutations were assessed in the representative formalin fixed paraffinembedded tissues (FFPETs) of 89 patients with GIST compared to 35 normal control (NC) subjects using the Real Time PCR (Rt-PCR) and immunohistochemistry (IHC).

Results: C-KIT mutations were detected in 61/89 (68.5%) patients, compared to none of the NC subjects (P=<0.001). Most mutations were detected in exon 11 (50.8%), followed by exon 9 (24.5%), exon 13 (19.6%), exon 7 (4.1%), and exon 12 (only 1%). C-KIT protein expression was detected in 69 (77.5%) patients compared to none of the NC subjects (p=<0.001). Significant measure of agreement was detected between C-KIT expression by IHC and and RT-PCR (K=0.774, p=0.0001).

The presence of C-KIT mutations associated significantly with large tumor size (P=0.01), High mitotic rate, lymph nodes metastasis and high tumor risk (p=0.001, for all). Patients with exon 11 mutations 26/31 (83.9%) had a better response to treatment compared to those with exon nine 7 (46.7%) and exon 13 mutation 1/12 (8.3%; P=0.001). Exon 11 mutations associated significantly with better PFS and OS compared to those with exon 9 and 13 mutations (P= 0.001, P= 0.0001; respectively). On univariate analysis, Exon 13 mutations was the most significantly associated with reduced PFS (P=0.0001), whereas advanced disease stage, absence of adequate safety margins and the site of exon mutation associated significantly with reduced OS. (P=0.012, P=0.05 and P=0.005; respectively.

Conclusion: GIST patients with C-KIT exon 11 mutations have a better response to imatinib treatment (predictive value), better OS and lower risk of disease progression than those with exon 9 and 13 mutations. Our specified mutations in exons 9& 13 mutations could be used as an independent prognostic factor for GIST patients.

Citation Format: Abeer A. Bahnnasy, Ola Khorshid, Mona S. Abdellateif, Ghada Y. Zakaria, Nasr M. Allahloubi, Auhood Nassar, Abdel-Rahman N. Zekri. Correlation between different C-KIT exon mutation and clinical outcome to Imatinib Mesylate treatment in patients with gastro-intestinal stromal tumors (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1777.
Abstract 1784: High expression of PTK-7 regulates the growth and invasion of triple-negative breast cancer cells
Authors: Bollu Lakshmi Reddy
Published: August 2019

Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis. Due to the frequent distant metastasis and lack of successful targeted therapies, the overall survival rate for the patients with TNBC is significantly lower than estrogen receptor positive and HER2 positive breast cancers. To identify potential druggable targets, previously, we performed a gene expression analysis and identified protein kinases that are highly expressed in ER-negative breast cancers. Through these studies, we discovered that the expression of pseudo kinase-7 (PTK-7) is highly elevated in TNBCs and correlated with p53 mutation status and disease progression. We hypothesize that high expression of PTK-7 is required for the growth and invasiveness of TNBC cells.

Experimental design and methods: In this study, we analyzed the expression status of PTK-7 in TNBC and the association of PTK-7 expression with p53 status and disease prognosis using publicly available datasets. Protein levels of PTK-7 were determined through western blotting analysis. The association of p53 and PTK-7 expression was determined using Oncomine analysis. Using specific siRNAs against PTK-7, we determined whether PTK-7 is required for the growth of TNBC cells. The role of PTK-7 in migration and invasion of TNBC cells was determined using Boyden trans-well assays.

Results: Analysis of publicly available datasets reveals that PTK-7 expression is elevated in TNBCs compared with normal breast and ER-positive breast cancers. High PTK-7 expression was found to be associated with p53 mutations. Knockdown of PTK-7 using siRNA significantly reduced the growth of TNBC cells in vitro. In vitrotrans-well migration and invasion studies also demonstrated that knockdown of PTK-7 significantly reduced the migration and invasion of TNBC cells. In contrast knockdown of PTK-7 did not affect the growth and invasiveness non-TNBC cells.

Conclusion: Our results suggest that the expression of PTK-7, a critical regulator of growth and invasion of TNBCs. These results suggest that PTK-7 is a promising target for the treatment of women with TNBC. This work was supported by Susan G Komen Promise Grant (PB), SAB Komen grant (PB) and Young Foundation grant (PB).

Citation Format: Lakshmi Reddy Bollu. High expression of PTK-7 regulates the growth and invasion of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1784.
Abstract 1791: RADIL regulates RAS downstream signaling
Authors: Choi Byeong Hyeok , Kou Ziyue , Philips Mark R. , Dai Wei
Published: August 2019

RAS proteins regulate a variety of cellular processes including cell survival, proliferation, and differentiation. RAS genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anti-cancer drugs in the clinic that target RAS proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate RAS activity or mediates its downstream signaling. Recently, we identified a series of proteins that are associated with HRAS using a combination of immunoprecipitation and mass spectrometry. We first focused on RADIL, a gene product with Ras-Association (RA) and DIL domains. RADIL is known to be a downstream effector of RAP1, inhibiting RhoA signaling in the regulation of cell survival, adhesion, and migration. We demonstrated that RADIL interacted with all three isoforms of RAS, including H, N, and KRAS. Interestingly, constitutively active HRAS (HRASG12V) was strongly associated with RADIL whereas dominant negative HRAS (HRASS17N) displayed a compromised association with RADIL, suggesting that RADIL may be required for RAS activation. We also found that Radil Knockout cells had weakened ERK phosphorylation whereas induced expression of RADIL promoted ERK phosphorylation, suggesting that RADIL is required for the RAS-MEK-ERK signaling cascade in vitro. Taken together, our current studies strongly suggest that RADIL may be important for regulating RAS signaling.

Citation Format: Byeong Hyeok Choi, Ziyue Kou, Mark R. Philips, Wei Dai. RADIL regulates RAS downstream signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1791.
Abstract 1821: LINC00346 regulates c-Myc transcriptional activity through CTCF in pancreatic cancer
Authors: Peng Wan-Xin , Mo Yin-Yuan
Published: August 2019

Pancreatic ductal adenocarcinoma (PDAC), a most challenging malignant disease, is estimated to become the second leading cause of cancer-related deaths in the United States between the year 2020 and 2030. Although the aberrant activation of KRAS has been demonstrated as the driving force of PDAC progression, the contribution of other molecules, especially long non-coding RNAs (lncRNAs), to PDAC progression remain largely unknown. In the present study, we interrogated the pancreatic adenocarcinoma dataset of the Cancer Genome Atlas (TCGA) consisting of 186 cases. Among 2,730 lncRNAs analyzed, LINC00346 revealed the highest alterations among these lncRNAs with 11% frequency at the RNA level. In addition, its upregulation was significantly associated with overall survival (OS) and disease-free survival (DFS), respectively. To determine the biological function of LINC00346 in pancreatic cancer, we next performed loss-of-function assay in pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 by CRISPR/Cpf1 technology using dual gRNA approach. The results showed that knockout (KO) of LINC00346 impaired pancreatic cancer cell proliferation, tumorigenesis, migration and invasion ability, while these phenotypes were restored by LINC00346 re-expression in KO cells (i.e., rescue experiment). Mechanistically, LINC00346 functions as a positive regulator of c-Myc. Further characterizations demonstrated that LINC00346 transcriptionally activates c-Myc expression by interaction with CTCF. Together, these results suggest that LINC00346 contributes to PDAC pathogenesis by activating c-Myc, and thus, LINC00346 may serve as a potential target for pancreatic cancer therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Wan-Xin Peng, Yin-Yuan Mo. LINC00346 regulates c-Myc transcriptional activity through CTCF in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1821.
Abstract 1823: SNORD78 promotes prostate cancer progression and regulates AR expression and signaling
Authors: Lombard Alan P. , Liu Chengfei , Armstrong Cameron M. , D'Abronzo Leandro S. , Lou Wei , Evans Christopher P. , Gao Allen C.
Published: August 2019

While small nucleolar RNAs (snoRNAs) have long been thought of as housekeeping genes, emerging evidence now suggests that snoRNAs are involved in more than previously thought and also in the development of disease. In cancer, it is understood that snoRNA expression and function can be altered, but little is known regarding the role of snoRNAs in these diseases. SNORD78 is a C/D box snoRNA which has been shown to be overexpressed in prostate cancer and associated with disease progression. However, there are no studies functionally characterizing SNORD78 in prostate cancer. In this study we sought to understand the functional contribution of SNORD78 in prostate cancer progression. Oncomine was used to assess SNORD78 expression in clinical prostate cancer samples. LNCaP and C4-2B prostate cancer cell lines were used. We used siRNAs to inhibit SNORD78 expression and cell viability assays to assess cell growth. ELISAs were used to assess PSA secretion and cell death. Western blots and quantitative PCR were used to assess cell death (PARP-cleavage) and AR expression and signaling. We found that SNORD78 expression was up-regulated in prostate tumors and associated with metastatic progression. Cell viability assays demonstrated that inhibition of SNORD78 expression resulted in a drastic reduction in prostate cancer cell growth and lower secretion of PSA into the media. Cell death ELISAs and western blots for cleaved-PARP showed that SNORD78 inhibition induced robust apoptosis in both LNCaP and C4-2B cells. As the androgen receptor (AR) is the primary target for prostate cancer therapy, we tested whether SNORD78 regulated AR expression and signaling. Inhibition of SNORD78 in both LNCaP and C4-2B cells resulted in reduced AR mRNA and protein levels. We also found that SNORD78 knockdown led to decreased levels of PSA and NKX3.1, suggesting that AR downstream signaling was inhibited. Our findings suggest that SNORD78 promotes prostate cancer cell viability and disease progression. We also demonstrate that SNORD78 regulates the AR and may be involved in promoting AR signaling needed for prostate cancer cell survival. Collectively, these data suggest that SNORD78 plays a critical role in prostate cancer progression and may represent a novel therapeutic target.

Citation Format: Alan P. Lombard, Chengfei Liu, Cameron M. Armstrong, Leandro S. D'Abronzo, Wei Lou, Christopher P. Evans, Allen C. Gao. SNORD78 promotes prostate cancer progression and regulates AR expression and signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1823.
Abstract 1824: Long noncoding RNA NR_027340 promotes head and neck cancer cell proliferation and glycolysis via PFKP
Authors: Yang Yifan , Wang Ru , Feng Ling , Shi Qian , Tan Chen , Fang Jugao
Published: August 2019

Introduction: Long noncoding RNAs (lncRNAs) play crucial roles in various cancers. Recent study has indicated that lncRNA NR_027340 was significantly upregulated in head and neck cancer (HNC). However, its multifaceted roles in HNC are unclear. Here we demonstrate the role of lncRNA NR_027340 in HNC cell proliferation and glycolysis.

Methods: qRT-PCR was used to detect lncRNA NR_027340 expression in HNC tissues, and the correlations between lncRNA NR_027340 and clinicopathologic features were then analyzed. RNA pull-down and mass spectrometry analysis were carried out to explore the lncRNA NR_027340-bound proteins. RNA immunoprecipitation (RIP) was used to confirm the association between lncRNA and relevant proteins. lncRNA NR_027340 knockout cell lines were generated using the CRISPR/Cas9. Colony formation, cell cycle analysis and flow cytometry were used to measure the proliferation of HNC cells. The production of lactate was observed to evaluate the glycolysis of HNC cells.

Results: We found lncRNA NR_027340 was significantly up-regulated in HNCs and the elevated lncRNA NR_027340 was correlated with poor clinical prognosis. Mechanistically, we identified PFKP as the protein partner of lncRNA NR_027340 in the cytoplasm. In addition, we found that both lncRNA NR_027340 and PFKP depletion reduced the cell proliferation and glycolysis in vitro. The ingenuity pathway analysis and Western blot assay indicated that lncRNA NR_027340 targeted PFKP to regulate cell proliferation and glycolysis in HNC.

Conclusion: These findings reveal a functional role for lncRNA NR_027340 in altering glycolysis and promoting proliferation in HNC cells, suggesting that lncRNA NR_027340 might serve as a prognostic and diagnostic indicator as well as a potential therapeutic target.

Citation Format: Yifan Yang, Ru Wang, Ling Feng, Qian Shi, Chen Tan, Jugao Fang. Long noncoding RNA NR_027340 promotes head and neck cancer cell proliferation and glycolysis via PFKP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1824.
Abstract 1830: Role of heat shock factor 1 (HSF1) on metabolic reprogramming of T -cells
Authors: Pandya Bhaumik D. , Mivechi Nahid M. , Moskophidis Dimitrios
Published: August 2019

Heat shock transcription factor 1 (HSF1) is normally activated in response to a variety of environmental stressors. Higher levels of HSF1 expression has been shown in many tumor types and this is associated with poor prognosis. Previous data from our laboratory indicate that genetic inactivation of HSF1 significantly delays ERBB2-induced mammary tumorigenesis and hepatocellular carcinoma induced by chemical carcinogen in mouse models. We have also shown that HSF1 is involved in controlling cellular bioenergetics. CD8-positive T cells plays an important role in anti-tumor immunity response. Our preliminary results show that genetic inactivation of HSF1 in T-cells leads to reduced oxygen consumption rate (OCR) as well as Extracellular Acidification Rate (ECAR). We have also observed that deletion of HSF1 from T cells delayed in-vitro activation of purified CD8-positive T cells upon stimulation with anti-CD3 and anti-CD28. Above characteristics corresponds to reduced cellular glycolysis and T cell proliferation. Further studies will be directed towards exploring the effects of HSF1 deletion in T cell activation induced anti-tumor immunity. This study is supported by the NCI grants; NCICA062130 and NCICA132640.

Citation Format: Bhaumik D. Pandya, Nahid M. Mivechi, Dimitrios Moskophidis. Role of heat shock factor 1 (HSF1) on metabolic reprogramming of T -cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1830.
Abstract 1865: Buserelin trisphenylmethanol derivatives as anti-cancer prodrugs
Authors: Beni Ryan , Boadi William , Antwi Renner
Published: August 2019

Buserelin (BUS) is a synthetic nonapeptide agonist analog of LH-RH, first reported by Hoechst in 1976. It is used for the treatment of advanced prostate cancer. Side effects of buserelin include musculoskeletal pain, hearing disorders, increased thirst, worsening hypertension, anxiety, memory and concentration disturbances, fatigue, nervousness, palpitations, leukopenia, thrombocytopenia, altered blood lipids, and glucose intolerance. Therefore, improving of the biological activity of BUS by increasing the cellular uptake and retention is a remedy to this end. In this research, tris(4-methoxyphenyl)methanol (TPM) derivatives were synthesized and linked to BUS through hydrophobic linkers to enhance and optimize the hydrophobicity of this drug with the expectation to improve the cellular uptake. In this regard, several BUS conjugates of TPM derivatives with optimized hydrophobicity were synthesized by the reaction of methoxy benzenes (e.g. anisole, 2-fluoroanisole, 2-trifluoromethylanisole, 2-methylanisole, or 1,2-dimethoxybenzene, and 1,3,5-trioxane, followed by the conjugation with BUS and lauric acid in the presence of HBTU/DIPEA/DIC in moderate yields. Comparative antiproliferative assays between TPM-BUS conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and BUS were performed against human acute lymphoblastic leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), and mouse pre adipocytes (3T3-L1) cells. TPM-BUS conjugates inhibited the cell proliferation of CCRF-CEM, SK-OV-3 and 3T3-L1 cells by 48-86%, 32-77%, 42-67%, respectively, at a concentration of 1-25 µM after 24-72 h of incubation. These data suggest that TPM-BUS derivatives with optimized hydrophobicity can be used to improve the biological activity of BUS.

Note: This abstract was not presented at the meeting.

Citation Format: Ryan Beni, William Boadi, Renner Antwi. Buserelin trisphenylmethanol derivatives as anti-cancer prodrugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1865.
Abstract 1870: Polyphenols from Korean artemisia annua L (pKAL) showed anti-cancer effects by multiple mechanisms on HCT116 human colorectal cancer cells
Authors: Lee Won Sup , Paramanantham Anjugam , Jeong Eun Joo , Raha Suchismita , Kim Hye Jung , Kim GonSup , Shin Sung Chul
Published: August 2019

Artemisia annua L., (Gaddongsook in Korean), an annual herb, has been used for a long time in Korean folk medicine for the treatment of infectious or chronic disorder In addition, it possesses anti-inflammatory, and anti-cancer activity. However, the molecular mechanism for anti-cancer properties of Korean Artemisia annua is still unclear. Here, we investigated the anti-cancer effects of we investigated the anti-cancer effects of polyphenols from Korean Artemisia annua (pKAL) on HCT116 human colon cancer cells. pKAL induced cell death associated with the alteration of cell morphology and DNA content. Annexin V and PI staining revealed that late phase apoptosis. pKAL induced ROS generation and the cell death was recovered by NAC. Western blot revealed that pKAL significantly up-regulated the gamma-H2AX, p21, p53, p62 and cleavage of caspases-3 and PARP in HCT116 cells. pKAL induced changes in the expression of p53-dependent targets (p53, p21, Bax, Bak, and Fas). It also induced alteration of the expression of autophagy-related proteins (beclin1, p62, LC3-I, and LC3-II) posttranslational modification of proteins (Lamin A/C, HSP90, GM130) involved in the maintenance of intracellular membrane integrity. Unexpectedly, the effect of pKAL on cell death pKAL was similar in both the P53 wild-type and p53-null HCT116 colon cancer cells, although there was some difference in cell cycle arrest; in P53 wild-type cells, pKAL appeared to induce G1 arrest at lower concentration (50 μg/mL), whereas in p53-null cells, pKAL induced G2/M arrest. The cell death was regulated by JNK and p38 MAPK signaling. The cancer effects of pKAL was similar in p53-null NCI-H1299 cells. We also investigated the protein changes that is related to cell death and cell integrity with anti-body array, which revealed that pKAL induced changes in NF-Kb, PI3K, Ras, TGF-beta signaling too. In conclusion, these findings suggest that pKAL induced anticancer effects by inducing late phase apoptotic cell death at least in part through p38 MAPK and JNK pathways in HCT 1116 human colon cancer cells. It also induced alteration of cell morphology mimic to necrosis in some cells with changes of proteins involved in the maintenance of intracellular membrane integrity. These effects was p53-independent.

Citation Format: Won Sup Lee, Anjugam Paramanantham, Eun Joo Jeong, Suchismita Raha, Hye Jung Kim, GonSup Kim, Sung Chul Shin. Polyphenols from Korean artemisia annua L (pKAL) showed anti-cancer effects by multiple mechanisms on HCT116 human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1870.
Abstract 1873: Water-pipe smoking induces epithelial-mesenchymal transition and enhances cell invasion of human breast cancer cells
Authors: Moustafa Ala-Eddin Al
Published: August 2019

Water-pipe smoking (WPS) has been recently recognized as an emerging risk factor for human health. The exact role of WPS in human breast cancer development and progression has not been investigated yet. We herein explored for the first time the impact of WPS on epithelial-mesenchymal transition (EMT) and cell invasion, which are considered crucial for cancer progression and metastasis in human carcinomas including breast. Our data revealed that WPS can initiate EMT in two human breast cancer cell lines, MCF-7 and BT20. WPS enhanced the cell invasion ability of both cell lines compared with their matched controls. We also found that WPS provoke a down- and up-regulation of two important regulator genes of cancer progression: E-cadherin and focal adhesion kinase (FAK). In addition, our data pointed out that WPS induces the activation of Erk1/Erk2 signaling pathways, which could be behind the stimulation of EMT and deregulation of E-cadherin and FAKgenes. Taken together, our results imply the significant effects of WPS on several cellular processes and genes involved in breast cancer progression and metastasis. Further studies are necessary to identify the clinical relevance and exact role of WPS in human carcinogenesis and metastasis. Key words: Water-pipe smoking, breast cancer, EMT, cell invasion, cell adhesion

Citation Format: Ala-Eddin Al Moustafa. Water-pipe smoking induces epithelial-mesenchymal transition and enhances cell invasion of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1873.
Abstract 1886: Zygote arrest 1 like (ZAR2): An EMT (epithelial-mesenchymal transition) modulator in breast cancer
Authors: Misra Smita , Singha Tanushree , Chaudhuri Gautam
Published: August 2019

Breast cancer is the most common cancer in women and distant site metastasis is the main cause of death in breast cancer patients. EMT is a natural phenomenon in development and wound healing. EMT is also one of the “Hallmarks of cancer”. EMT is involved in tumor cell progression, invasion, and metastasis. During the process of EMT, epithelial cancer cells acquire molecular alternations that facilitate the loss of epithelial features and gain of mesenchymal phenotype. Such transformation promotes cancer cell migration and invasion. Decrease in E-cadherin expression and gain of N-cadherin and vimentin expression is indicative of EMT phenotype. Working with our recently characterized C4 zinc finger containing RNA binding protein ZAR2 we observed that levels of ZAR2 are significantly lower in the invasive breast cancer cells compared to non-invasive cells. Knockdown of ZAR2 in the non-invasive breast cancer cells increased the in-vitro invasiveness of these cells whereas forced expression of ZAR2 in the invasive breast cancer cells reduced their invasiveness. To understand the possible mechanism of ZAR2-mediated regulation of invasiveness of the breast cancer cells we studied differential gene expression in the ZAR2 knocked down non-invasive breast cancer cells by RNA-seq analysis. One of the genes that are significantly elevated in the ZAR2 knocked down cells is the invasion determining enzyme ATP6V0A4 along with ERBIN and ABCA12. Here we present our observation regarding ZAR2 regulation of ATP6V0A4 and its effect on metastasis and invasion potential of the breast cancer cells. Supported in part by NIH grant 1U54RR026140, Meharry Bridge and 2U54MD007586-32Revised from Meharry RCMI Program in Women’s Health funds to SM and DOD grants BC990678 and BC050641 to GC.

Citation Format: Smita Misra, Tanushree Singha, Gautam Chaudhuri. Zygote arrest 1 like (ZAR2): An EMT (epithelial-mesenchymal transition) modulator in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1886.