Synthetic Lethal Interactions between EGFR and PARP Inhibition in Human Triple Negative Breast Cancer Cells

Few therapeutic options exist for the highly aggressive triple negative breast cancers (TNBCs). In this study, we report that a contextual synthetic lethality can be achieved both in vitro and in vivo with combined EGFR and PARP inhibition with lapatinib and ABT-888, respectively. The mechanism involves a transient DNA double strand break repair deficit induced by lapatinib and subsequent activation of the intrinsic pathway of apoptosis. Further dissection of the mechanism reveals that EGFR and BRCA1 can be found in the same protein complex, which is reduced by lapatinib. Interestingly, lapatinib also increases cytosolic BRCA1 and EGFR, away from their nuclear DNA repair substrates. Taken together, these results reveal a novel regulation of homologous recombination repair involving EGFR and BRCA1 interaction and alteration of subcellular localization. Additionally, a contextual synthetic lethality may exist between combined EGFR and PARP inhibitors.     

MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells

Introduction

Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method

In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results

MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions

MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.     

乳腺癌相关的细胞内信号通路

乳腺癌是女性中常见的恶性肿瘤之一.乳腺癌的发生、发展、转移及耐药性的产生与细胞内的信号通路密切相关,其中雌激素受体(estrogen receptor,ER)信号通路、胰岛素样生长因子受体（insulin-like growth factor receptor,IGFR)信号通路和表皮生长因子受体（epidermal growth factor receptor,EGFR）信号通路尤为重要．深入了解ER、IGFR和EGFR三条信号通路的作用机制及它们之间的交叉对话对于寻找新的更有效的肿瘤治疗靶点至关重要．本文综述了近年来有关ER、IGFR和EGFR三条信号通路研究进展及这三条通路与乳腺癌关系．     

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

Background

Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.

Materials and Methods

PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.

Results

PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.

Conclusions

Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.     

Alternative Signaling Pathways as Potential Therapeutic Targets for Overcoming EGFR and c-Met Inhibitor Resistance in Non-Small Cell Lung Cancer

The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC₅₀ concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.     

Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen

Aim

Triple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light.

Method

After validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 after injection and 100 J/cm² of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm² on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm² on day 2, which were performed two times every week (“two split” NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2, which were performed three times per week (“three split” NIR-PIT).

Result

Both specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, “two split” NIR-PIT; p < 0.01, “three split” NIR-PIT; p < 0.001) compared with control groups.

Conclusion

NIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.     

Sevoflurane Modulates AKT Isoforms in Triple Negative Breast Cancer Cells. An Experimental Study

(1) Background: Triple negative breast cancer (TNBC) is a highly aggressive tumor, associated with high rates of early distant recurrence and short survival times, and treatment may require surgery, and thus anesthesia. The effects of anesthetic drugs on cancer progression are under scrutiny, but published data are controversial, and the involved mechanisms unclear. Anesthetic agents have been shown to modulate several molecular cascades, including PI3K/AKT/mTOR. AKT isoforms are frequently amplified in various malignant tumors and associated with malignant cell survival, proliferation and invasion. Their activation is often observed in human cancers and is associated with decreased survival rate. Certain anesthetics are known to affect hypoxia cell signaling mechanisms by upregulating hypoxia-inducible factors (HIFs). (2) Methods: MCF-10A and MDA-MB 231 cells were cultivated and CellTiter-Blue^® Cell Viability assay, 2D and 3D matrigel assay, immunofluorescence assays and gene expressions assay were performed after exposure to different sevoflurane concentrations. (3) Results: Sevoflurane exposure of TNBC cells results in morphological and behavioral changes. Sevoflurane differently influences the AKT isoforms expression in a time-dependent manner, with an important early AKT3 upregulation. The most significant effects occur at 72 h after 2 mM sevoflurane treatment and consist in increased viability, proliferation and aggressiveness and increased vimentin and HIF expression. (4) Conclusions: Sevoflurane exposure during surgery may contribute to cancer recurrence via AKT3 induced epithelial–mesenchymal transition (EMT) and by all three AKT isoforms enhanced cancer cell survival and proliferation.     

肿瘤干细胞生长相关的信号转导通路

肿瘤干细胞是肿瘤中存在的一小群具有自我更新和分化潜能的细胞,也是存在于肿瘤 组织中具有干细胞样能力的肿瘤细胞亚群,在肿瘤的发生、发展中起着非常重要的作用.近年来发现,肿瘤干细胞的生长调控与Wnt、Notch、Hedgehog等多种信号转导通 路有关.本文简要综述了肿瘤干细胞生长相关信号转导通路的研究进展,旨在为肿瘤干细胞研究和临床应用提供理论依据.     

Determination of the Effects of Bee Venom on Triple Negative Breast Cancer Cells in Vitro

Honeybees provide multiple products such as bee venom (BV) which are used for various nutritional and medicinal purposes. BV has received great attention due to its wide range of bioactive components with potential anti-cancer effects on different cancers. Triple negative breast cancer (TNBC) is defined as an aggressive type of breast cancer and new therapeutic targets are required for its treatment. In the current literature information is varied about the composition and quantity of BV bioactive compounds as well as the origin of BV and its significance. In this context, the cytotoxic and apoptotic effects of BV with a higher rate of mellitin from Apis mellifera anatoliaca (Muğla ecotype) on MDA-MB-231 cells was evaluated, in vitro. The cytotoxic, apoptotic and morphological effects of BV were determined by WST-1, Annexin V, cell cycle analysis and Acridine Orange staining. The results showed that BV caused apoptotic cell death in TNBC cells at a lower dose (0.47 μg/mL, p<0.01). This study suggests that BV could be developed as a potential therapeutic agent for cancer treatment. However, the mechanism of BV-induced apoptosis death should be clarified at the molecular level.     

Fibulin-5在三阴型乳腺癌细胞侵袭中的功能研究

的：研究Fibulin-5蛋白在三阴性乳腺癌（TNBC）m胞转移中的作用。方法：以三阴性乳腺癌细胞系（MDA-MB-231）分化而来的三株高中低不同转移能力的子细胞系为模型,高表达和抑制Fibulin．5在其中的表达,研究其对细胞侵袭能力的影响。结果：三阴性乳腺癌细胞内本底的Fibulin．5与其转移能力正相关,另外上调和抑制Fibulin-5可以改变其原有的侵袭能力。结论：Fibulin-5在三阴性乳腺癌细胞转移中发挥重要作用,抑制Fibulin-5可以降低三阴性乳腺癌细胞的侵袭能力。     

Spectrum of EGFR Gene Copy Number Changes and KRAS Gene Mutation Status in Korean Triple Negative Breast Cancer Patients

Anti-epidermal growth factor receptor (EGFR) therapy has been tried in triple negative breast cancer (TNBC) patients without evaluation of molecular and clinical predictors in several randomized clinical studies. Only fewer than 20% of metastatic TNBCs showed response to anti-EGFR therapy. In order to increase the overall response rate, first step would be to classify TNBC into good or poor responders according to oncogenic mutation profiles. This study provides the molecular characteristics of TNBCs including EGFR gene copy number changes and mutation status of EGFR and KRAS gene in Korean TNBC patients. Mutation analysis for EGFR, KRAS, BRAF and TP53 from a total of 105 TNBC tissue samples was performed by direct sequencing, peptide nucleic acid-mediated PCR clamping method and real-time PCR. Copy number changes of EGFR gene were evaluated using multiplex ligation-dependent probe amplification. Out of all 105 TNBCs, 15.2% (16/105) showed EGFR copy number changes. Among them, increased or decreased EGFR copy number was detected in 13 (5 single copy gain, 2 amplification and 4 high-copy number amplification) and 3 cases (3 hemizygous deletion), respectively. The mutation frequencies of KRAS, EGFR and TP53 gene were 1.9% (G12V and G12D), 1.0% (exon 19 del) and 31.4%, respectively. There was no BRAF V600E mutation found. Future studies are needed to evaluate the clinical outcomes of TNBC patients who undergo anti-EGFR therapy according to the genetic status of EGFR.     

癌、干细胞和癌干细胞共同拥有的信号传导通路

干细胞（SC）是具有无限自我更新和分化能力的细胞,随着对SC研究的不断深入,人们发现SC与肿瘤细胞有许多共性,如无限增生能力、迁移能力及在某些条件下能相互转化,故提出了肿瘤起源于SC的学说。探讨癌、SC和癌干细胞（CSC）之间可能存在的共同信号传导通路,以便发现治疗CSC的靶位。     

miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*

目的: 探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法: 利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果: TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论: miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。     

Antiproliferative and Apoptotic Effects of pH-Responsive Veratric Acid-Loaded Polydopamine Nanoparticles in Human Triple Negative Breast Cancer Cells

Veratric acid (VA) is plant-derived phenolic acid known for its therapeutic potential, but its anticancer effect on highly invasive triple-negative breast cancer (TNBC) is yet to be evaluated. Polydopamine nanoparticles (nPDAs) were chosen as the drug carrier to overcome VA's hydrophobic nature and ensure a sustained release of VA. We prepared pH-sensitive nano-formulations of VA-loaded nPDAs and subjected them to physicochemical characterization and in vitro drug release studies, followed by cell viability and apoptotic assays on TNBC cells (MDA-MB-231 cells). The SEM and zeta analysis revealed spherical nPDAs were uniform size distribution and good colloidal stability. In vitro drug release from VA-nPDAs was sustained, prolonged and pH-sensitive, which could benefit tumor cell targeting. MTT and cell viability assays showed that VA-nPDAs (IC50=17.6 μM) are more antiproliferative towards MDA-MB-231 cells than free VA (IC50=437.89 μM). The induction of early and late apoptosis by VA-nPDAs in the cancer cells was identified using annexin V and dead cell assay. Thus, the pH response and sustained release of VA from nPDAs showed the potential to enter the cell, inhibit cell proliferation, and induce apoptosis in human breast cancer cells, indicating the anticancer potential of VA.     

肿瘤干细胞表面标志物及主要信号通路

肿瘤干细胞是存在于肿瘤组织中的具有自我更新、增殖、分化的部分细胞群,对肿瘤的发生、发展有十分重要的作用. 肿瘤干细胞特异的表面分子及其异常活化的信号通路,是其区别于其他肿瘤细胞的特性.寻找和鉴定特异的肿瘤干细胞的表面标志物,从而识别肿瘤组织中的肿瘤干细胞,并进行相关信号调控机制研究,是肿瘤早期诊断及肿瘤干细胞靶向治疗的关键. 本文简要概述了肿瘤干细胞相关的表面标志物及信号通路的研究进展,旨在为进一步开展针对肿瘤干细胞的抗体靶向治疗提供新思路.     

GALNT14 Involves the Regulation of Multidrug Resistance in Breast Cancer Cells

GALNT14 is a member of N-acetylgalactosaminyltransferase enzyme family and mediates breast cancer cell development. Here, we find that GALNT14 regulates multidrug resistance (MDR) in breast cancer. The expression of GALNT14 is associated with MDR in breast cancer. Higher level of GALNT14 facilitates MCF-7 cells to resist Adriamycin, whereas knockdown of GALNT14 sensitizes cells to Adriamycin. Moreover, the expression of GALNT14 associates with the expression of P-gp, the efflux pump localized on the cell membrane, which could be the underlying mechanism of how GALNT14 induces MDR. In-depth analysis shows that GALNT14 regulates the stability of P-gp. Finally, GALNT14 associates with higher level of P-gp in chemotherapy-resistant human breast cancer tissues. Taken together, our studies reveal a molecular mechanism in breast cancer MDR.     

RB1 Status in Triple Negative Breast Cancer Cells Dictates Response to Radiation Treatment and Selective Therapeutic Drugs

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1^met, fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA⁺/CD24^−/low/CD44⁺ cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.