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PDF file - 45K, Relationship of CXCR4 expression and clinicopathological characteristics of gallbladder carcinoma

PDF file, 1016KB, Supplementary Figure 1. Immunohistochemical staining shows L1CAM is expressed in the invasive areas of ATC. Cases 1 through 3 have L1CAM expression in the invasive islands (red arrowhead). Cases 7 and 8 express L1CAM in the invasive fronts of ATC (black arrowhead). Supplementary Figure 2. Aberrant L1CAM expression is observed in recurrent ATC. Case 4 has negative L1CAM expression at the PTC tumor (left). Black arrowhead indicates the internal positive control at the peripheral nerve bundles that has normal L1CAM expression. However, the recurred ATC tumor exhibits positive L1CAM expression in Case 4 (right). Supplementary Figure 3. Immunohistochemical staining of cyclin D1 and p53 in ATC (Original magnification: �200). Supplementary Figure 4. L1CAM expression in primary cultured follicular cells (PCFC), primary cultured PTC (PCPTC), PTC cell lines (TPC1 and BCPAP), and ATC cell lines (FRO and 8505C). The ovarian carcinoma cell line SKOV3 is used as a positive control. Thyroglobulin is a marker of thyroid-derived cells.

PDF file - 299K, Supplementary Figure 1: Enhanced migration and invasion of GBC cells overexpressing CXCL12. SNU-308 cells were stably transfected with CXCL12 as described in Materials and Methods. Migration (A) and invasion (B) assays were performed as described in Materials and Methods. Three independent experiments were performed in duplicate. Data are expressed as mean � SD; **p < 0.01 versus mock.; Supplementary Figure 2. The effect of CXCR7 on the CXCL12-induced growth, migration, and invasion of GBC cells. SNU-308 cells were transiently transfected with control siRNA or CXCR7 siRNA for 48 hours. Proliferation (A), migration (B), and invasion (C) assays were performed as described in Materials and Methods. Three independent experiments were performed in duplicate. Data are expressed as mean � SD; **p

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Purpose: Although recent studies have suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) is important in the progression of various malignancies, its role in gallbladder carcinoma (GBC) remains unknown. We investigated CXCL12 expression in GBC and its biologic and prognostic role in GBC tumorigenesis.Experimental Design: We examined CXCL12 expression in tumor specimens from 72 patients with GBC by immunohistochemistry and analyzed the correlation between CXCL12 expression and clinicopathologic factors or survival. The functional significance of CXCL12 expression was investigated by CXCL12 treatment and suppression of CXCR4, a major receptor of CXCL12, as well as by CXCL12 overexpression in in vitro and in vivo studies.Results: CXCL12 was differentially expressed in GBC tissues. CXCL12 expression was significantly associated with a high histologic grade (P = 0.042) and nodal metastasis (P = 0.015). Multivariate analyses showed that CXCL12 expression (HR, 8.675; P = 0.014) was an independent risk factor for patient survival. CXCL12 significantly increased anchorage-dependent and -independent growth, migration, invasion, adhesiveness, and survival of GBC cells in vitro, and these effects were dependent on CXCR4. Consistent with these results, overexpression of CXCL12 significantly promoted GBC tumorigenicity in a xenograft model.Conclusions: Our results indicate that GBC cells express both CXCL12 and its receptor CXCR4, and CXCL12 may have a role in GBC progression through an autocrine mechanism. In addition, CXCL12 is a novel independent poor prognostic factor in patients with GBCs. Thus, targeting CXCL12 and CXCR4 may provide a novel therapeutic strategy for GBC treatment. Clin Cancer Res; 18(12); 3270–80. ©2012 AACR.

Purpose: We describe herein a novel P447_L455 deletion in the C2 domain of PIK3CA in a patient with an ER+ breast cancer with an excellent response to the PI3Kα inhibitor alpelisib. Although PIK3CA deletions are relatively rare, a significant portion of deletions cluster within amino acids 446–460 of the C2 domain, suggesting these residues are critical for p110α function. Experimental Design: A computational structural model of PIK3CAdelP447-L455 in complex with the p85 regulatory subunit and MCF10A cells expressing PIK3CAdelP447-L455 and PIK3CAH450_P458del were used to understand the phenotype of C2 domain deletions. Results: Computational modeling revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Coimmunoprecipitation experiments showed reduced binding of the C2 deletion mutants with p85 compared with wild-type p110α. The MCF10A cells expressing PIK3CA C2 deletions exhibited growth factor–independent growth, an invasive phenotype, and higher phosphorylation of AKT, ERK, and S6 compared with parental MCF10A cells. All these changes were ablated by alpelisib treatment. Conclusions: C2 domain deletions in PIK3CA generate PI3K dependence and should be considered biomarkers of sensitivity to PI3K inhibitors. Clin Cancer Res; 24(6); 1426–35. ©2017 AACR.

Background: Activating mutations in ERBB2 (HER2) are found in ~2% of solid tumors, including ~5% of metastatic breast cancers. The spectrum of HER2 mutations varies according to tumor type, with kinase domain (KD) missense mutations being most common in breast cancer, while exon 20 insertions are most common in non-small cell lung cancer (NSCLC). Breast cancer cells engineered with activating HER2 mutations show elevated HER2 phosphorylation and enhanced oncogenic growth. Activating mutations of other receptor tyrosine kinases such as EGFR affect receptor internalization, but whether HER2 mutations alter receptor internalization is not yet known. In addition, clinical responses to HER2 antibody-drug conjugates (ADCs) like T-DM1 have been noted in HER2-mutant NSCLC, raising the possibility that HER2 mutations alter receptor internalization, thus enhancing ADC sensitivity. Therefore, we aimed to 1) determine whether mutant HER2 receptor internalization is altered, and 2) determine whether altered internalization contributes to HER2 ADC sensitivity in HER2-mutant breast cancer. Methods: HER2 subcellular localization was investigated by immunofluorescence (IF) in MCF7 cells where HER2L755S, HER2V777L, or HER2L869R (or HER2WT) have been ‘knocked-in' using AAV-mediated recombination. We used ELISA-based endocytosis assays to quantify the internalization efficiency of trastuzumab-labeled HER2 in cell lines with WT and mutant HER2. Cell viability upon treatment with T-DM1 was tested using conventional 2D and 3D growth assays. Results: Partial colocalization of HER2 with clathrin, measured by IF, was observed in MCF7 cells, which was not affected by HER2 mutations. In the endocytosis assay, trastuzumab was inefficiently internalized over a period of 4 hours. In MCF7 or MCF10A breast epithelial cells, introduction of HER2L755S, HER2V777L, or HER2L869R did not significantly affect internalization following trastuzumab labeling, or after stimulation with EGF or neuregulin. In line with these results, MCF7 cells expressing HER2L755S, HER2V777L, or HER2L869R were not sensitive to T-DM1 in 2D cell viability assays or 3D Matrigel assays. In contrast, HER2 internalization was significantly more efficient in BT474 cells with wild-type HER2 amplification, which was accompanied by exquisite sensitivity of BT474 cells to T-DM1 both in vitro and in vivo. HER2 endocytic efficiency was significantly increased in MCF10A cells ectopically expressing HER2S310F, a mutation in the extracellular domain, or HER2A775>YVMA, a common insertion mutation in NSCLC. Conclusion: Breast cancer cells expressing HER2 KD missense mutations do not exhibit enhanced HER2 internalization and are not sensitive to T-DM1. However, cells with the HER2S310F mutation and the HER2A775>YVMA insertion showed enhanced HER2 endocytosis, consistent with clinical responses to T-DM1 in NSCLC patients with these mutations. Citation Format: Hiroaki Akamatsu, Rosa Mino, Marcel Mettlen, Dan Ye, Dhivya R. Sudhan, Albert Lin, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Sumanta Chatterjee, Sandra L. Schmid, Carlos L. Arteaga, Ariella B. Hanker. HER2 missense mutations in breast cancer cells do not alter HER2 internalization or sensitivity to T-DM1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3675.

Although the 17q23 amplicon has been associated with luminal B breast cancer (BC) and high risk of recurrence, a specific gene or genes in this region that would be causal to endocrine resistance have not yet been uncovered. We performed whole transcriptome analysis on RNA extracted from 58 estrogen receptor (ER)+ BCs treated with neoadjuvant letrozole for median 7.2 months. PRR11 (Proline rich 11), located in 17q23, was upregulated in non-responding tumors as defined by relapse after a median follow up of 5 years and/or a preoperative endocrine prognostic index (PEPI) ≥4. Differential gene expression analysis between tumors expressing low vs high PRR11 mRNA showed that BC signatures associated with proliferation, IGF-1 and PI3K signaling were enriched in tumors with high PRR11 expression. Rate of PRR11 amplification is 15.2% in the Metastatic Breast Cancer project, but 9.5% and 9.4% in METABRIC and The Cancer Genome Atlas (TCGA), respectively. Gene Set Enrichment Analysis revealed an enrichment of hallmark gene sets associated with proliferation in PRR11-amplified ER+ BCs in METABRIC and TCGA. Integrated analysis of gene expression with on-treatment Ki67 levels from three independent studies with operable ER+ BCs treated with neoadjuvant aromatase inhibitor (ACOSOG-Z1031, NCT00651976, Llombart-Cussac et al.) showed that PRR11 was the only gene in 17q23 with a significant correlation with a high Ki67 levels across all studies. PRR11 knockdown inhibited E2-independent growth of HCC1428 LTED (long-term estrogen deprived) and MCF7 LTED cells in culture and MCF7 xenografts. PRR11 siRNA also inhibited growth of fulvestrant-resistant and tamoxifen-resistant MCF7 cells. Conversely, PRR11 transduction induced MDA-MB-134VI cell growth under estrogen-depleted conditions. Using a PCR array with 84-cell cycle genes, we identified SKP2, CDKN1A, CCNB2, CCNA2, CKS2 and CCNB1 as genes downregulated by PRR11 knockdown. Except for SKP2 and CDKN1A, expression of all those genes was elevated in PRR11-amplifiedER+ BCs in TCGA and METABRIC. Suggesting a link to activation of PI3K signaling, we found the proline-rich motif of PRR11 associates with the SH3 domain of the p85 regulatory subunit of PI3K. We hypothesized that this association suppresses p85 homodimer formation, thus facilitating binding of PI3Kα (p110α)-p85 dimers to IRS1, retention of p110α at the plasma membrane and, hence, activation of PI3K/AKT. To test this, we co-transfected HEK293T cells with HA-p85 and FLAG-p85. Forced expression of PRR11 reduced HA-p85 and FLAG-p85 homodimers as shown by HA and FLAG pulldowns followed by FLAG and HA immunoblots, respectively. PRR11 overexpression enhanced insulin-stimulated association of IRS1 to p110α and activation of AKT. PRR11 knockdown reduced insulin/IGF-1/2-stimulated p-AKT. In METABRIC and TCGA, PRR11 amplification and PIK3CA mutations are exclusive of each other, suggesting these alterations would be functionally linked with the same pathway. Connectivity map analysis with the list of genes significantly overexpressed in ER+/PRR11-amplified BCs predicted PI3K inhibitors as perturbations that suppress such gene list. In the MGH/Sanger dataset, PRR11-amplified BC cell lines displayed significantly higher sensitivity to the pan-PI3K inhibitor pictilisib compared to cell lines without PRR11 amplification. Finally, inhibition of PI3Kα by siRNA or alpelisib abrogated E2-independent growth and insulin-stimulated growth of PRR11-transduced MDA-MB-134VI and MCF10A cells, respectively, suggesting p110α is required for the growth promoting effects of PRR11. These data suggest that 1) PRR11 is a mediator of resistance to antiestrogens via amplification of PI3K/AKT signaling, and 2) PI3Kα is a potential therapeutic target in ER+ BCs harboring PRR11 amplification. Citation Format: Kyung-min Lee, Angel Guerrero-Zotano, Ariella Hanker, Alberto Servetto, Dhivya Sudhan, Luigi Formisano, Valerie Jansen, Paula González-Ericsson, Melinda Sanders, Thomas Stricker, Lewis Cantley, Carlos Arteaga. A neoadjuvant trial with letrozole identifies PRR11 in the 17q23 amplicon as a mechanism of resistance to endocrine therapy in ER-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS6-06.

Background: FGFR1 amplification occurs in about 15% of estrogen receptor-positive (ER+) breast cancers and is associated with resistance to endocrine therapy. In these tumors, nuclear FGFR1 has been shown to interact with ERα and alter gene expression through binding to chromatin. However, the mechanisms underpinning nuclear FGFR1-mediated gene transcription remain unclear. Thus, we sought to elucidate mechanisms to explain the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer.Results: FGFR1 ChIP-Seq detected 4408 DNA binding sites in CAMA1 ER+/FGFR1-amplified breast cancer cells cultured in estrogen-free conditions; 67% of these sites were enriched at promoter regions, suggesting a role of FGFR1 in gene transcription regulation. ChIP-qPCR assay confirmed FGFR1 binding to promoter regions of genes such as CCND1, MYC, VEGFA, JUNB and SMAD5 in both CAMA1 and MDA-MB-134 ER+/FGFR1-amplified cells and also in an ER+/FGFR1-amplified patient derived xenograft (HCI-011). RNA-Seq of CAMA1 cells revealed that expression of FGFR1-bound genes was substantially higher than non FGFR1-bound genes (p0.25), representing those whose expression is likely regulated by FGFR1. This high signature score was associated with worse disease free survival (DFS; 263.7 months vs not reached; HR=1.72, CI 1.39-2.12; p Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Kyung-min Lee, Dhivya R Sudhan, Ariella B Hanker, Sumanta Chatterjee, Albert Lin, Saurabh Mendiratta, Nicholas James, Ralf Kittler, Carlos L Arteaga. Fibroblast growth factor receptor 1 associates with promoters genome-wide and regulates gene transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-04.

Purpose: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR–mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer. Experimental Design: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay. Results: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model. Conclusions: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization. Clin Cancer Res; 19(16); 4335–46. ©2013 AACR.

Cytotoxic chemotherapy is the standard of care for patients with triple negative breast cancer (TNBC). Most patients with advanced TNBC progress after chemotherapy and die from metastatic disease. MCL1 is an anti-apoptotic Bcl-2 family member known to sequester and inactivate pro-apoptotic Bcl-2 family proteins and, thus, contribute to chemotherapy resistance. We previously reported that ~45% of residual TNBCs that remain in the breast after neoadjuvant chemotherapy harbor MCL1 amplification, suggesting a causal role for MCL1 in drug resistance. A recent report (Goodwin et al. 2015) suggested that siRNA-mediated ablation of MCL1 does not induce apoptosis in claudin-low TNBC cells with a cancer stem cell (CSC) gene expression signature. CSCs comprise a rare population of cells with tumor-initiating properties and refractoriness to chemotherapy. In this study, we showed that MCL1 expression is elevated in claudin-low TNBC SUM159PT and MDA436 CSCs as measured by ALDH+ by flow cytometry and ability to form mammospheres. RNA interference of MCL1 in SUM159PT cells reduced CSCs and attenuated tumor formation in vivo. Mitochondrial oxidative phosphorylation (mtOXPHOS) plays a crucial role in maintenance of CSCs. MCL1 has been shown to localize in the mitochondrial matrix and contribute to mitochondrial respiration. Thus, we hypothesized that MCL1 contributes to enrichment of TNBC CSCs and chemotherapy resistance via mitochondrial regulation. Stable transfection and overexpression of MCL1 in MDA468 cells increased oxygen consumption ratio, mitochondrial membrane potential, and production of reactive oxygen species (ROS), all features of activated mtOXPHOS. Conversely, RNAi-mediated ablation of MCL1 in SUM159PT and MDA436 cells repressed these markers of activated mtOXPHOS. A mutant of MCL1 lacking its mitochondrial target sequences (MTS) was unable to localize in mitochondria and, when transfected into MDA468 cells, reduced the CD44high/CD24low fraction and mammosphere formation. We next tested VU0659158, a BH3 mimetic in development at Vanderbilt that disrupts MCL1 interactions with BH3 domain-containing proteins, such as BID, BIM, NOXA and PUMA. Treatment of SUM159PT cells with VU0659158 increased caspase activity but did not attenuate mammosphere formation. Analysis of mRNA expression in TCGA revealed that genes induced by mtOXPHOS involved in the hypoxia pathway are significantly up-regulated in MCL1 amplified breast cancers. Finally, pharmacological inhibition of HIF-1α, a key regulator of hypoxia, with digoxin decreased CSCs and attenuated tumor formation in vivo. These data suggest that 1) MCL1 confers resistance to chemotherapy by expanding CSCs via mtOXPHOS independent of its BH3 domain-mediated, anti-apoptotic function, and 2) targeting mitochondrial respiration and the hypoxia pathway may delay or reverse chemotherapy resistance in MCL1 amplified TNBC. Citation Format: Kyung-min Lee, Jennifer Giltnane, Justin Balko, Luis Schwarz, Angel Guerrero, Katie Hutchinson, Mellissa Hicks, Violeta Sanchez, Melinda Sanders, Taekyu Lee, Edward Olejniczak, Stephen Fesik, Carlos Arteaga. Mitochondrial MCL1 maintains triple negative breast cancer stem cells and contributes to chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2017-3890

Triple negative breast cancer is the most virulent subtype of this malignancy. Although initially responsive to cytotoxic chemotherapy, most patients with TNBC eventually develop drug-resistant disease. We previously reported co-amplification of MYC and MCL1 in a cohort of TNBCs after neoadjuvant chemotherapy (Balko et al. Cancer Discov. 2014). In addition, MYC and MCL1 are overexpressed in paclitaxel-resistant compared to paclitaxel-sensitive TNBC cells. We hypothesized that MYC and MCL1 play a role in chemotherapy resistance. TNBC cell lines with claudin-low gene expression exhibited the highest levels of MYC and MCL1 mRNAs. This molecular subtype of breast cancer is enriched for cancer stem cells (CSCs) and exhibits resistance to neoadjuvant chemotherapy. Knockdown of both MYC and MCL1 by siRNA in SUM159PT and MDA-MB-436 TNBC cells significantly reduced mammosphere formation and CSC markers such as ALDH and CD44high/CD24low. Conversely, overexpression of ectopic MYC and MCL1 in MDA-MB-468 cells increased mammosphere formation and CSC markers. It has been reported that mitochondrial oxidative phosphorylation (OXPHOS) is enhanced in CSCs. Thus, we investigated a role for MYC and MCL1 in mitochondrial OXPHOS using Seahorse XFe extracellular flux analyzer. CSCs with high expression of MYC and MCL1 sorted by flow cytometry exhibited increased mitochondrial OXPHOS compared to non-CSCs. Transfection of MYC and MCL1 expression vectors into MDA-MB-468 cells enhanced mitochondrial OXPHOS activity. Conversely, knockdown of MYC and MCL1 in SUM159PT and MDA-MB-436 cells reduced mitochondrial OXPHOS activity. MYC mediated these effects by promoting mitochondrial biogenesis whereas MCL1 potentiated mitochondrial OXPHOS via localization into the mitochondrial matrix, independent of its ability to associate with BH3-domain containing anti-apoptotic proteins (BAD, BCL2, BCL-XL). Deletion of the mitochondrial target sequence (MTS) of MCL1 impaired its ability to increase mitochondrial OXPHOS and mammosphere formation. Reactive oxygen species (ROS), which are produced in CSCs as a by-product of activated mitochondrial OXPHOS, were abrogated by MYC and MCL1 siRNA. To determine the effect of ROS on CSCs, we treated SUM159PT and MDA-MB-436 cells with hydrogen peroxide (H2O2). Treatment with H2O2 enriched CSCs, which showed up-regulation of several cancer stem cell genes such as Nanog and IL-8. Enrichment of CSCs by H2O2 was diminished by n-acetylcysteine (NAC), an antioxidant. These data suggest that MYC and MCL1 co-amplification confers drug resistance to TNBC via mitochondrial OXPHOS-mediated enrichment of CSCs. Moreover, MYC and MCL1 stimulate H2O2 production, which potentiates CSCs. Thus, targeting mitochondrial OXPHOS and H2O2 may be an effect strategy to delay or reverse resistance to chemotherapy in MYC and MCL1 amplified TNBC. Citation Format: Kyung-min Lee, Jennifer Giltnane, Justin Balko, Carlos Arteaga. MYC and MCL1 cooperatively promote chemotherapy-resistant cancer stem cells through regulation of mitochondrial biogenesis and oxidative phosphorylation. [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 3328.

Summary: Breast cancer is a heterogeneous type of cancer accompanying extensive immune cell infiltration. Although its mortality rate has been decreasing with early diagnosis and introduction of molecular targeted therapies, it is still one of the world leading cause of cancer death in women. In breast cancer, molecular subtyping is used for the patient stratification and treatment decisions, emphasizing the importance of inter-patient tumoral heterogeneity in the tumor cell behavior. Tumor cells within a patient may also manifest intra-tumoral heterogeneity, which may cause treatment resistance. Single cell gene expression profiling would reveal both inter- and intra-tumoral heterogeneity in breast cancer encompassing tumor cells and associated stromal and immune cells. Experimental Process: From 4 different subtype primary breast cancer and 2 lymph node metastases, single cells were captured and amplified through C1™Single-cell Auto Prep System (Fluidigm) with the SMARTer Ultra Low RNA Kit. Sequencing libraries were constructed with the Nextera XT DNA sample Prep Kit and sequenced through a Hiseq2500 (Illumina) as 100-bp paired end mode of the TruSeq Rapid PE Cluster kit and Truseq Rapid SBS kit. Computational Process: RNA sequencing reads were aligned to the modified human genome reference using 2-pass mode of STAR_2.4.0b, and transcript per million (TPM) values were obtained as the relative gene expression levels of single cells through RSEM v1.2.17. Results: As most breast cancers originate from the mammary epithelium, we distinguished carcinoma cells from associated non-tumor cells by epithelial cell adhesion molecule (EPCAM) signature genes. We validated the distinction with chromosomal gene expression patterns and gene set enrichment analyses using tumor, stromal, or immune genesets. With or without removal of non-tumor cells, TNBC tumor cells showed the highest gene expression heterogeneity, recapitulating the known inter-patient heterogeneity. TNBC tumor cells from a single patient were categorized into 6 different TNBC-subtypes, further demonstrating intra-tumoral heterogeneity. In addition, immune cells identified in the TNBC tumor were naïve B cells and regulatory T cells, suggesting non-activated status of the immune system. Conclusions: The high resolution gene expression profiling revealed features of breast cancer transcriptome which may provide clues for molecular directed therapies targeting the tumor or the immune compartment. Citation Format: Woosung Chung, Hye Hyeon Eum, Kyu-Tae Kim, Kyung-Min Lee, Arum Jo, WonShik Han, Hae-Ock Lee, Woong-Yang Park. Identification of the carcinoma and immune cells in the breast cancer by single-cell RNA sequencing. [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 160.

Retinoblastoma is the most common intraocular malignant tumor in children, affecting approximately 1 in 20,000 live births. In this study, we investigated the role of L1, a transmembrane protein, in proliferation and chemoresistance of retinoblastoma to figure out the potential for the novel therapeutic target. Retinoblastoma tissues demonstrated varying degrees of L1 positivity in 86.6% (26/30) of samples. In particular, the degree of L1 positivity was inversely related with that of Flexner-Wintersteiner rosette formation. Further in vitro studies using stable cell lines which showed up- or down-regulation of L1 demonstrated that L1 was associated with cell-cell adhesion and proliferation of retinoblastoma cells. Retinoblastoma cells with low expression of L1 showed decreased tumor formation in vivo. In addition, L1 expression was related with resistance to carboplatin, one of the most-widely utilized chemotherapeutic agents against retinoblastoma. In line with these results, retinoblastoma cells with higher expression of L1 demonstrated increased resistance to carboplatin in vivo. We also observed diffuse and dense expression of L1 in retinoblastoma tissues from 4 patients who underwent enucleation despite intensive chemotherapy based on carboplatin. Taken together, L1 was related with proliferation and chemoresistance of retinoblastoma and might be a potential therapeutic target of retinoblastoma. Citation Format: Dong Hyun Jo, Kyungmin Lee, Jin Hyoung Kim, Hyoung Oh Jun, Young Hoon Kim, Young-Lai Cho, Young Suk Yu, Jeong-Ki Min, Jeong Hun Kim. The role of L1 in proliferation and chemoresistance of retinoblastoma. [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 2469.