Expression of HER-2/neu in oral squamous cell carcinoma

Background: HER-2/neu is a member of the human epidermal growth factor (HER) family of transmembrane tyrosine kinases, which is significantly associated with the pathogenesis of various cancer types. The aim was to evaluate the expression of HER-2/neu in oral squamous cell carcinoma (OSCC) as a potential biomarker to target antigens for specific immunotherapy in OSCC.Methods: One hundred and forty histologically diagnosed OSCC cases were identified. Four to five-micrometer thick formalin-fixed, paraffin-embedded tumor sections were stained with Haematoxylin and Eosin (H and E). Histological grade was assessed according to WHO/Broders classification, while tumors were staged according to the American Joint Committee on Cancer (AJCC) TNM classification from stage I to IV. Immunohistochemistry was performed by using Rabbit monoclonal antibody against HER-2/neu (EP700Y, cell marquee and diluted 1:50). FISH was performed on positive cases using Vysis PathVysion HER-2 DNA probe (Abbott USA). Probes consist of LSI HER gene spectrum orange and control probe CEP 17 spectrum green.Results: In this study, males were mostly effected (64.3%) with buccal mucosa (49%) to be the commonly involved site for OSCC. Majority of cases were moderately differentiated (62.1%) and 50.7% tumors were Stage IV. HER-2/neu was found to be positive (2+) in one case of OSCC, however weak to moderate complete membrane staining was observed in \u3e10% of the tumor cells. One hundred and thirty nine cases were HER-2/neu negative. FISH analysis of HER-2/neu positive cases also showed gene amplification (Her2-neu/ CEp 17 = 225/33 = 7.2).Conclusions: The study showed disparity in the expression of HER-2/neu in OSCC, which is due to multiple reasons. Therefore therapy against HER-2/neu in OSCC is debatable

Clinicopathological and prognostic significance of mitogen-activated protein kinases (MAPK) in breast cancers

Background Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. Methods Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray. Moreover, reverse phase protein array was applied to quantify protein expression of MAPKs in six breast cancer cell lines with different phenotypes including HER2-transfected cells. Results MAPKs expression was associated with clinicopathological variables characteristic of good prognosis. These associations were most significant in the whole series and in the ER? subgroup compared to other BC classes. Most of MAPKs showed a positive association with ER, BCL2 and better outcome and were negatively associated with the proliferation marker Ki67 and p53. Association of MAPK with HER2 was mainly seen in the ER- subgroup. Reverse phase protein array confirmed immunohistochemistry results and revealed differential expression of MAPK proteins in ER? and ER- cell lines. Conclusions MAPKs are associated with good prognosis and their expression is mainly related to ER. Studying a large panel rather than individual biomarkers may provide improved understanding of the pathwa

ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC

Background: We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized.Methods: We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients.Results: Overexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 andc-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients’ outcome was independent of tumor grade, stage and size, and ER status.Conclusion: ADA3 overexpression enhances cell proliferation that is associated with increased expression of c-MYC. Expression patterns with respect to ADA3/c-MYC can divide patients into four significantly different subgroups, with c-MYC High and ADA3 Low status independently predicting poor survival in patients

Targeting PARP1 in XRCC1-deficient sporadic invasive breast cancer or preinvasive ductal carcinoma in situ induces synthetic lethality and chemoprevention

© 2018 American Association for Cancer Research. Targeting PARP1 for synthetic lethality is a new strategy for breast cancers harboring germline mutations in BRCA. However, these mutations are rare, and reactivation of BRCA-mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting more common sporadic breast cancers and preinvasive ductal carcinoma in situ (DCIS) are desirable. Here we show that downregulation of XRCC1, which interacts with PARP1 and coordinates base excision repair, is an early event in human breast cancer pathogenesis. XRCC1-deficient DCIS were aggressive and associated with increased risk of local recurrence. Human invasive breast cancers deficient in XRCC1 and expressing high PARP1 levels also manifested aggressive features and poor outcome. The PARP1 inhibitor olaparib was synthetically lethal in XRCC1-deficient DCIS and invasive breast cancer cells. We conclude that targeting PARP1 is an attractive strategy for synthetic lethality and chemoprevention in XRCC1-deficient breast cancers, including preinvasive DCIS. Significance: These findings show that loss of XRCC1, which is associated with more malignant DCIS, can be exploited by PARP inhibition, suggesting its application as a promising therapeutic and chemoprevention strategy in XRCC1-deficient tumor cells

EHD1 is Required for IGF-1R-mediated Oncogenic Signaling in Ewing Sarcoma

Background and Significance: Ewing Sarcoma (EWS) is the second most common malignant bone tumor of children and adolescents. Patients with metastatic or recurrent disease have very poor outcomes. The receptor tyrosine kinase(RTK) insulin-like-growth-factor-1-receptor (IGF-1R) is upregulated in 93% of EWS patients with anti-IGF-1R antibodies and kinase inhibitors in clinical studies. However, with only ~10% of patients achieving objective responses, delineation of novel pathways that facilitate IGF-1R-driven oncogenesis in EWS could provide avenues for more effective therapy. The RTK levels and compartmentalization at the cell surface determine their access to growth factors, thus dictating the downstream oncogenic signaling. Our lab has demonstrated that EPS15-homology-domain-containing-protein-1 (EHD1) regulates traffic of cell surface receptors, including RTKs. We observed high frequency (67%) of EHD1 overexpression in 266 primary EWS patient tumor tissues, and Kaplan-Meier survival analysis of publicly available mRNA expression data showed that high EHD1 expression was associated with shorter patient survival. Objective/Question: This study aims to comprehend the underlying role of EHD1 in EWS oncogenesis. Experimental design and Results: In both dox-inducible EHD1-shRNA knockdown and EHD1-CRISPR-Cas9-knockout (KO) EWS cell line models(TC71, A673, and SKES1), we observed a significant impairment of in vitro oncogenic properties namely, cell proliferation, migration, invasion, soft-agar colony formation, and tumor-sphere formation, and the phenotypes were restored upon mouse-EHD1 rescue. Furthermore, by orthotopically implanting TC71 cells in the tibia of nude mice(xenograft model), we demonstrated a significant reduction in tumor size upon EHD1-depletion. Using a phospho-RTK profiling antibody array, we found reduced phospho-IGF-1R levels upon EHD1-KD, identifying IGF-1R as a potential target of regulation by EHD1. EHD1-KO reduced surface IGF-1R levels under steady-state and ligand-free conditions in EWS cells. IGF-1R and EHD1 were also found to colocalize intracellularly and co-immunoprecipitate after IGF-1 stimulation. Notably, EHD1-KO impaired the IGF-1R-mediated activation of downstream AKT and MAPK pathways. Mechanistically, EHD1 was shown to regulate traffic of newly synthesized IGF-1R and recycled pools from the Golgi to the cell surface, and in absence of EHD1, intracellular IGF-1R was shunted to the lysosome resulting in degradation. Finally, by dual targeting of EHD1 (genetic depletion) and IGF-1R (small-molecule-inhibitor Linsitinib), we observed an additive effect on inhibition of EWS cell proliferation and migration and upregulation of apoptosis. Conclusions: Our studies indicate a novel regulatory pathway of EHD1 requirement in IGF-1R cell surface display and sustaining IGF-1R-mediated oncogenesis in EWS. This highlights the prospects of therapeutic co-targeting of EHD1 and IGF-1R, thus enhancing IGF-1R targeted therapies in EWS.https://digitalcommons.unmc.edu/chri_forum/1040/thumbnail.jp

Mammalian alteration/deficiency in activation 3 (Ada3) is essential for embryonic development and cell cycle progression.

Ada3 protein is an essential component of histone acetyl transferase containing coactivator complexes conserved from yeast to human. We show here that germline deletion of Ada3 in mouse is embryonic lethal, and adenovirus-Cre mediated conditional deletion of Ada3 in Ada3(FL/FL) mouse embryonic fibroblasts leads to a severe proliferation defect which was rescued by ectopic expression of human Ada3. A delay in G(1) to S phase of cell cycle was also seen that was due to accumulation of Cdk inhibitor p27 which was an indirect effect of c-myc gene transcription control by Ada3. We further showed that this defect could be partially reverted by knocking down p27. Additionally, drastic changes in global histone acetylation and changes in global gene expression were observed in microarray analyses upon loss of Ada3. Lastly, formation of abnormal nuclei, mitotic defects and delay in G(2)/M to G(1) transition was seen in Ada3 deleted cells. Taken together, we provide evidence for a critical role of Ada3 in embryogenesis and cell cycle progression as an essential component of HAT complex

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients.

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients\u27 outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients

Upregulation of Cyclin B2 (CCNB2) in breast cancer contributes to the development of lymphovascular invasion

Lymphovascular invasion (LVI) is a key step in breast cancer (BC) metastasis. Targeting the molecular drivers of LVI can improve BC patients’ management. However, the underlying molecular mechanisms of LVI are complex and interconnected with various carcinogenesis pathways. This study aimed to identify the key regulatory gene associated with LVI and to investigate its mechanisms of action and prognostic significance. Artificial neural network (ANN) was applied to two large transcriptomic datasets of BC with well-characterised LVI status. Cyclin B2 (CCNB2) was identified in the top genes associated with LVI positivity. In vitro functional assays were carried out to assess the role of CCNB2 in tumour cell behaviour and their interactions with endothelial cells using a panel of BC cell lines. Large annotated BC cohorts were used to assess the clinical and prognostic role of CCNB2 at the transcriptomic and protein levels. Knockdown (KD) of CCNB2 mRNA reduced BC cell migration, inhibited proliferation, blocked the G2/M transition during the cell cycle and increased the number of apoptotic cells. Importantly, KD of CCNB2 reduced BC cell lines adherence and transmigration across endothelial cell lines. High CCNB2 protein expression was independently associated with LVI positivity in addition to other features of aggressive behaviour, including larger tumour size, higher histological grade, hormonal receptor-negativity, and HER2-positivity, and with shorter survival. We conclude that CCNB2 plays a crucial role in LVI development in BC, implying that CCNB2 could confer a promising therapeutic target to inhibit LVI and reduce metastatic events

Blocking c-MET/ERBB1 Axis Prevents Brain Metastasis in ERBB2+ Breast Cancer

Brain metastasis (BrM) remains a significant cause of cancer-related mortality in epidermal growth factor receptor 2-positive (ERBB2+) breast cancer (BC) patients. We proposed here that a combination treatment of irreversible tyrosine kinase inhibitor neratinib (NER) and the c-MET inhibitor cabozantinib (CBZ) could prevent brain metastasis. To address this, we first tested the combination treatment of NER and CBZ in the brain-seeking ERBB2+ cell lines SKBrM3 and JIMT-1-BR3, and in ERBB2+ organoids that expressed the c-MET/ERBB1 axis. Next, we developed and characterized an orthotopic mouse model of spontaneous BrM and evaluated the therapeutic effect of CBZ and NER in vivo. The combination treatment of NER and CBZ significantly inhibited proliferation and migration in ERBB2+ cell lines and reduced the organoid growth in vitro. Mechanistically, the combination treatment of NER and CBZ substantially inhibited ERK activation downstream of the c-MET/ERBB1 axis. Orthotopically implanted SKBrM3+ cells formed primary tumor in the mammary fat pad and spontaneously metastasized to the brain and other distant organs. Combination treatment with NER and CBZ inhibited primary tumor growth and predominantly prevented BrM. In conclusion, the orthotopic model of spontaneous BrM is clinically relevant, and the combination therapy of NER and CBZ might be a useful approach to prevent BrM in BC