Nonphosphorylatable PEA15 mutant inhibits epithelial-mesenchymal transition in triple-negative breast cancer partly through the regulation of IL-8 expression.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood. METHODS: We established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis. RESULTS: We found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1. CONCLUSIONS: Our results show that PEA15 phosphorylation status serves as an important regulator for PEA15's dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC.

Anti-tumor and anti-metastasis efficacy of E6201, a MEK1 inhibitor, in preclinical models of triple-negative breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) lacks the receptor targets estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and thus, it does not respond to receptor-targeted treatments. TNBC has higher recurrence, metastasis, and mortality rates than other subtypes of breast cancer. Mounting data suggest that the MAPK (also known as RAS-RAF-MEK-ERK) pathway is an important therapeutic target in TNBC. METHODS: To evaluate anti-tumor and anti-metastasis efficacy of E6201, we used cell proliferation assay, soft agar assay, cell cycle assay, Annexin V staining assay, immunoblotting analysis, immunohistochemistry, migration assay, invasion assay, mammary fat pad xenograft, and experimental and spontaneous metastasis xenograft models. We also evaluated the anti-tumor efficacy of E6201 plus CDK4/6 inhibitor, mTOR inhibitor, or ATR inhibitor. RESULTS: E6201 inhibited TNBC cell colony formation, migration, and invasion in a dose-dependent manner. E6201 induced G1 cell cycle arrest and apoptosis. E6201 inhibited TNBC xenograft growth and inhibited TNBC lung metastasis and improved mouse survival in experimental metastasis and spontaneous metastasis assays. Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung and decreased phosphorylated ERK expression in a dose-dependent manner. Combination of E6201 with CDK4/6 inhibitor or mTOR inhibitor enhanced E6201's in vitro anti-tumor efficacy. CONCLUSION: These results indicate that E6201 exhibits anti-tumor efficacy against TNBC in vitro and anti-metastasis efficacy against TNBC in vivo. These results provide a rationale for further clinical development of E6201 as a MAPK-pathway-targeted therapy for TNBC.

Correction to: Anti-tumor and anti-metastasis efficacy of E6201, a MEK1 inhibitor, in preclinical models of triple-negative breast cancer.

Unfortunately in the original publication of the article, the author's funding support has been mentioned incorrectly. The correct funding statement should read as "This work was supported by the Morgan Welch Inflammatory Breast Cancer Research Program, the State of Texas Rare and Aggressive Breast Cancer Research Program, MD Anderson's Cancer Center Support Grant (P30CA016672, used the Characterized Cell Line Core Facility and Flow Cytometry and Cellular Imaging Facility), and Spirita Oncology, LLC."The first affiliations was incorrect in the original article. The correct information is given below.

Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth.

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.

CD44 expression contributes to trastuzumab resistance in HER2-positive breast cancer cells.

Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. CD44, a putative breast cancer stem cell (CSC) marker, is overexpressed in trastuzumab-resistant breast cancer cells. While CSC-related genes may play a role in the development of trastuzumab resistance, conflicting results have been published about CSC response to trastuzumab. We hypothesized that CD44 contributes to trastuzumab resistance independently of its role as a CSC marker. We used trastuzumab-sensitive breast cancer cell lines and their trastuzumab-resistant isogenic counterparts to evaluate the role of CD44 in response to trastuzumab. miRNA and mRNA expression were analyzed using microarray chips. A gene set enrichment analysis was created and matched with response to trastuzumab in cells and patient samples. The proportions of CSC in trastuzumab-resistant cells were similar to or lower than in the trastuzumab-sensitive cells. However, CD44 expression levels were significantly higher in both trastuzumab-resistant cell lines and its knockdown led to an increased response to trastuzumab. The invasiveness and anchorage-independent growth of trastuzumab-resistant cells were higher and blocked by downregulation of CD44. Results also showed that CD44-related resistance to trastuzumab is regulated by miRNAs. We identified a CD44-related gene expression profile that correlated with response to trastuzumab in cell lines and breast cancer patients. CD44 mediates trastuzumab resistance in HER2-positive breast cancer cells independently of its role as a CSC marker and that this role of CD44 is partly regulated by miRNA.

A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in HER2-overexpressing breast cancer cells through FOXO3-mediated Bim1 expression.

Although there are effective HER2-targeted agents, novel combination strategies in HER2-overexpressing breast cancers are needed for patients whose tumors develop drug resistance. To develop new therapeutic strategy, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/EGFR dual tyrosine kinase inhibitor, in HER2+ breast cancer cells. We assessed the combinational synergistic effect and its mechanism by CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time PCR, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in two xenograft mouse models (BT474 and SUM190, P < 0.001). The synergistic anti-tumor activity of the entinostat/lapatinib combination was due to downregulation of phosphorylated Akt, which activated transcriptional activity of FOXO3, resulting in induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistance-HER2-overexpressing cells to the trastuzumab/lapatinib combination and enhanced the anti-proliferation effect compare with single or double combination treatment. This study provides evidence that entinostat has enhanced anti-tumor effect in combination with HER2-targeted reagent, lapatinib, and resulting in induction of apoptosis by FOXO3-mediated Bim1 expression. Our finding justifies for conducting a clinical trial of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2-overexpressing breast cancer resistant to trastuzumab-based treatment.

Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSC), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. However, the role of MELK in TNBC metastasis is unknown; we sought to address this in the current study. We found that MELK mRNA levels were higher in TNBC tumors [8.11 (3.79-10.95)] than in HR+HER2- tumors [6.54 (2.90-9.26)]; P < 0.001]. In univariate analysis, patients with breast cancer with high-MELK-expressing tumors had worse overall survival (P < 0.001) and distant metastasis-free survival (P < 0.01) than patients with low-MELK-expressing tumors. In a multicovariate Cox regression model, high MELK expression was associated with shorter overall survival after adjusting for other baseline risk factors. MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition, and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells (P < 0.05). Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice (P < 0.001). Our findings indicate that MELK supports metastasis by promoting epithelial-to-mesenchymal transition and the CSC phenotype in TNBC. Significance: These findings indicate that MELK is a driver of aggressiveness and metastasis in TNBC.

High ERK protein expression levels correlate with shorter survival in triple-negative breast cancer patients.

The mitogen-activated protein kinase (MAPK) signaling pathway is known to be activated in triple-negative breast cancer (TNBC). Extracellular signal-related kinase (ERK), a member of the MAPK pathway, promotes cell proliferation, angiogenesis, cell differentiation, and cell survival. To assess the prognostic impact of ERK in TNBC patients, relative quantities of ERK (ERK-2 and pMAPK) and direct targets of the ERK pathway (MAPK/ERK kinase 1, phospho-enriched protein in astrocytes [PEA]-15, phosphorylated (p)PEA-15, tuberous sclerosis protein 2, p70S6 kinase, and p27) were measured using reverse-phase protein arrays in tumor tissue from patients with TNBC (n = 97) and non-TNBC (n = 223). Protein levels in patients with TNBC were correlated with clinical and tumor characteristics and outcome. The median age of patients with TNBC was 55 years (range, 27-86 years). Disease stage was I in 21%, II in 60%, and III in 20% of the patients. In a multivariate analysis, among patients with TNBC, those with ERK-2-overexpressing tumors had a lower overall survival rate than those with low ERK-2-expressing tumors (hazard ratio [HR], 2.76; 95% confidence interval [CI], 1.19-6.41). However, high pMAPK levels were associated with a significantly higher relapse-free survival rate (HR, 0.66; 95% CI, 0.46-0.95). In conclusion, ERK-2 and pMAPK are valuable prognostic markers in TNBC. Further studies are justified to elucidate ERK's role in TNBC tumorigenicity and metastasis.

Role of epidermal growth factor receptor in breast cancer.

Decades of research in molecular oncology have brought about promising new therapies which are designed to target specific molecules which promote tumor growth and survival. The epidermal growth factor receptor (EGFR) is one of the first identified important targets of these novel antitumor agents. Approximately half of cases of triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) overexpress EGFR. Thus, EGFR inhibitors for treatment of breast cancer have been evaluated in several studies. However, results so far have been disappointing. One of the reasons for these unexpected results is the lack of biomarkers for predicting which patients are most likely to respond to EGFR inhibitors. Recent studies have shown that EGFR and its downstream pathway regulate epithelial-mesenchymal transition, migration, and tumor invasion and that high EGFR expression is an independent predictor of poor prognosis in IBC. Further, recent studies have shown that targeting EGFR enhances the chemosensitivity of TNBC cells by rewiring apoptotic signaling networks in TNBC. These studies indicate that EGFR-targeted therapy might have a promising role in TNBC and IBC. Further studies of the role of EGFR in TNBC and IBC are needed to better understand the best way to use EGFR-targeted therapy-e.g., as a chemosensitizer or to prevent metastases-to treat these aggressive diseases.

Prognostic impact of phosphorylated HER-2 in HER-2+ primary breast cancer.

PURPOSE: Tyrosine 1248 is one of the autophosphorylation sites of human epidermal growth factor receptor (HER)-2. We determined the prognostic value of the expression level of tyrosine 1248-phosphorylated HER-2 (pHER-2) in patients with HER-2(+) primary breast cancer using a reverse-phase protein array. PATIENTS AND METHODS: The optimal cutoff value of pHER-2 for segregating disease-free survival (DFS) was determined by receiver operating characteristic (ROC) curve analysis. Five-year DFS for pHER-2 expression level was estimated with the Kaplan-Meier method using both derivation (n = 162) and validation (n = 227) cohorts. RESULTS: Of the 162 patients in the derivation cohort, 26 had high HER-2 expression levels. The area under the ROC curve for pHER-2 level and DFS was 0.662. Nineteen of the 162 patients (11.7%) had high pHER-2 expression levels (pHER-2(high)); 143 patients (88.3%) had low pHER-2 expression levels (pHER-2(low)). Among the 26 patients with high HER-2 expression levels, the 17 pHER-2(high) patients had a significantly lower 5-year DFS rate than the nine pHER-2(low) patients (23.5% versus 77.8%). On multivariate analysis, only pHER-2(high) independently predicted DFS in the Cox proportional hazards model. In the validation cohort, among 61 patients with high HER-2 expression, the difference in 5-year DFS rates between pHER-2(high) (n = 7) and pHER-2(low) (n = 54) patients was marginal (57.1% versus 81.5%). CONCLUSION: In patients with HER-2(+) primary breast cancer, pHER-2(high) patients had a lower 5-year DFS rate than pHER-2(low) patients. Quantification of pHER-2 expression level may provide prognostic information beyond the current standard HER-2 status.

Novel mechanism of reduced proliferation in ovarian clear cell carcinoma cells: cytoplasmic sequestration of CDK2 by p27.

OBJECTIVE: Ovarian clear cell carcinoma (CCC) carries a poor prognosis because of its insensitivity to chemotherapy. We previously found an association between reduced proliferation of CCC and chemoresistance; here we investigated the mechanism of the reduced proliferation. METHODS: We assessed cell cycle function by measuring the activity of cyclin-dependent kinases (CDKs) and the protein expression of cyclins, the CDK inhibitors, and p53 in 22 ovarian cancer cell lines and 60 human ovarian cancer specimens. We examined the cellular location of p27, p27 phosphorylated at threonine 157 (p27(Thr157)), and CDK2 protein by confocal microscopy and western blotting. We tested the effect of the inhibitor of phosphatidylinositol-3-kinase (PI3K) and small interfering RNA against p27 (si-p27) in two CCC cell lines (RMG-I, SMOV-2). RESULTS: CCC cells had lower CDK2 activity and higher p27 expression than serous adenocarcinoma (SA) cells. Low CDK2 activity correlated with high p27 protein expression. p27(Thr157) sequestered CDK2 in the cytoplasm, but PI3K inhibitor or si-p27 maintained CDK2 in the nucleus and restored its activity. In human specimens, CDK2 was mostly in the cytoplasm and was spatially associated with p27; CDK2 activity was lower in the CCC than in the SA specimens. si-p27 enhanced the cytotoxic effect of cisplatin, doxorubicin, and gemcitabine in both RMG-I cells and SMOV-2 cells. CONCLUSIONS: Reduced CDK2 activity via the cytoplasmic sequestration of CDK2 by p27(Thr157) may contribute to suppression of CCC proliferation. A prospective study is needed to determine whether the cytoplasmic sequestration of CDK2 results in the chemoresistance of CCC.

Birinapant Enhances Gemcitabine's Antitumor Efficacy in Triple-Negative Breast Cancer by Inducing Intrinsic Pathway-Dependent Apoptosis.

Triple-negative breast cancer (TNBC) is the most aggressive subgroup of breast cancer, and patients with TNBC have few therapeutic options. Apoptosis resistance is a hallmark of human cancer, and apoptosis regulators have been targeted for drug development for cancer treatment. One class of apoptosis regulators is the inhibitors of apoptosis proteins (IAPs). Dysregulated IAP expression has been reported in many cancers, including breast cancer, and has been shown to be responsible for resistance to chemotherapy. Therefore, IAPs have become attractive molecular targets for cancer treatment. Here, we first investigated the antitumor efficacy of birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspases (SMACs), in TNBC. We found that birinapant as a single agent has differential antiproliferation effects in TNBC cells. We next assessed whether birinapant has a synergistic effect with commonly used anticancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), erlotinib (EGFR inhibitor), and gemcitabine, in TNBC. Among these tested drugs, gemcitabine showed a strong synergistic effect with birinapant. Birinapant significantly enhanced the antitumor activity of gemcitabine in TNBC both in vitro and in xenograft mouse models through activation of the intrinsic apoptosis pathway via degradation of cIAP2 and XIAP, leading to apoptotic cell death. Our findings demonstrate the therapeutic potential of birinapant to enhance the antitumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins.

Expression of phosphoprotein enriched in astrocytes 15 kDa (PEA-15) in astrocytic tumors: a novel approach of correlating malignancy grade and prognosis.

Phosphoprotein enriched in astrocytes 15 kDa (PEA-15) is a multifunctional protein that was first identified in brain astrocytes and that has subsequently been shown to be expressed in different tissues. Despite its many important roles, the clinical significance of PEA-15 expression levels in astrocytic tumors has yet to be properly defined. We studied the PEA-15 expression pattern of 65 patients [diagnosed according to World Health Organization (WHO) criteria] with diffuse astrocytoma (WHO grade II), anaplastic astrocytoma (grade III), and glioblastoma (grade IV). PEA-15 expression levels were immunohistochemically measured and categorized as no, low, or high expression. All tumors expressed PEA-15 in our study. Twenty-three (35.4%) and 42 (64.6%) tumors expressed low and high PEA-15 levels, respectively. In grade II astrocytoma (diffuse astrocytoma) and grade III astrocytoma (anaplastic astrocytoma), 100% and 88.9% of patients expressed high PEA-15 levels, respectively, while a smaller number (50%) of patients with grade IV astrocytoma (glioblastoma) expressed high PEA-15 levels. PEA-15 expression level was inversely associated with WHO grade (P = 0.0006). Next, we evaluated prognosis and PEA-15 expression levels in 43 patients with high-grade astrocytomas based on the following parameters: age, gender, WHO grade, surgical resection extent, MIB-1 labeling index (LI), and PEA-15 expression level. Multivariable analyses revealed that high PEA-15 expression level displayed a significant correlation with longer overall survival (OS) in high-grade astrocytomas (P = 0.0024). Patients with total resection survived significantly longer (P = 0.0044) than those with lower resection extent, while patients with MIB-1 labeling index ≤25% indicated significant (P = 0.0434) correlation with OS as well. In conclusion, PEA-15 expression level was inversely associated with WHO grade and may serve as an important prognostic factor for high-grade astrocytomas.

Non-Phosphorylatable PEA-15 Sensitises SKOV-3 Ovarian Cancer Cells to Cisplatin.

The efficacy of cisplatin-based chemotherapy in ovarian cancer is often limited by the development of drug resistance. In most ovarian cancer cells, cisplatin activates extracellular signal-regulated kinase1/2 (ERK1/2) signalling. Phosphoprotein enriched in astrocytes (PEA-15) is a ubiquitously expressed protein, capable of sequestering ERK1/2 in the cytoplasm and inhibiting cell proliferation. This and other functions of PEA-15 are regulated by its phosphorylation status. In this study, the relevance of PEA-15 phosphorylation state for cisplatin sensitivity of ovarian carcinoma cells was examined. The results of MTT-assays indicated that overexpression of PEA-15AA (a non-phosphorylatable variant) sensitised SKOV-3 cells to cisplatin. Phosphomimetic PEA-15DD did not affect cell sensitivity to the drug. While PEA-15DD facilitates nuclear translocation of activated ERK1/2, PEA-15AA acts to sequester the kinase in the cytoplasm as shown by Western blot. Microarray data indicated deregulation of thirteen genes in PEA-15AA-transfected cells compared to non-transfected or PEA-15DD-transfected variants. Data derived from The Cancer Genome Atlas (TCGA) showed that the expression of seven of these genes including EGR1 (early growth response protein 1) and FLNA (filamin A) significantly correlated with the therapy outcome in cisplatin-treated cancer patients. Further analysis indicated the relevance of nuclear factor erythroid 2related factor 2/antioxidant response element (Nrf2/ARE) signalling for the favourable effect of PEA-15AA on cisplatin sensitivity. The results warrant further evaluation of the PEA-15 phosphorylation status as a potential candidate biomarker of response to cisplatin-based chemotherapy.

Differential functions of ERK1 and ERK2 in lung metastasis processes in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer characterized by metastasis, drug resistance and high rates of recurrence. With a lack or targeted therapies, TNBC is challenging to treat and carries a poor prognosis. Patients with TNBC tumors expressing high levels of ERK2 have a poorer prognosis than those with low ERK2-expressing tumors. The MAPK pathway is often found to be highly activated in TNBC, however the precise functions of the ERK isoforms (ERK1 and ERK2) in cancer progression have not been well defined. We hypothesized that ERK2, but not ERK1, promotes the cancer stem cell (CSC) phenotype and metastasis in TNBC. Stable knockdown clones of the ERK1 and ERK2 isoforms were generated in SUM149 and BT549 TNBC cells using shRNA lentiviral vectors. ERK2 knockdown significantly inhibited anchorage-independent colony formation and mammosphere formation, indicating compromised self-renewal capacity. This effect correlated with a reduction in migration and invasion. SCID-beige mice injected via the tail vein with ERK clones were employed to determine metastatic potential. SUM149 shERK2 cells had a significantly lower lung metastatic burden than control mice or mice injected with SUM149 shERK1 cells. The Affymetrix HGU133plus2 microarray platform was employed to identify gene expression changes in ERK isoform knockdown clones. Comparison of gene expression levels between SUM149 cells with ERK2 or ERK1 knockdown revealed differential and in some cases opposite effects on mRNA expression levels. Those changes associated with ERK2 knockdown predominantly altered regulation of CSCs and metastasis. Our findings indicate that ERK2 promotes metastasis and the CSC phenotype in TNBC.

Acquired resistance to erlotinib in A-431 epidermoid cancer cells requires down-regulation of MMAC1/PTEN and up-regulation of phosphorylated Akt.

Erlotinib (Tarceva), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has clinical activity in advanced lung cancer, but disease that initially responds to erlotinib eventually progresses. The mechanism of this acquired resistance is unclear. We established two erlotinib-resistant pools of A-431 cells, a well-characterized epidermoid cancer cell line that constitutively overexpresses EGFR and is sensitive to erlotinib, by continuous exposure to erlotinib over a 6-month period. The extent of EGFR gene amplification or mutation of the EGFR tyrosine kinase domain was not altered in the resistant cells. Intracellular erlotinib concentrations, determined by liquid chromatography-tandem mass spectrometry, were almost the same in all three cell lines. Immunoprecipitation with EGFR antibody followed by detection with phosphotyrosine antibody revealed that erlotinib effectively reduced EGFR phosphorylation in both parental cells and resistant cells. Erlotinib induced mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) and suppressed phosphorylated Akt (Ser(473)) but not in the erlotinib-resistant cells. Overexpression of MMAC1/PTEN by transfection with Ad.MMAC1/PTEN or by pharmacologic suppression of Akt activity restored erlotinib sensitivity in both resistant pools. Further, transfection of parental A-431 cells with constitutively active Akt was sufficient to cause resistance to erlotinib. We propose that acquired erlotinib resistance associated with MMAC1/PTEN down-regulation and Akt activation could be overcome by inhibitors of signaling through the phosphatidylinositol 3-kinase pathway.

Activity of lapatinib is independent of EGFR expression level in HER2-overexpressing breast cancer cells.

Epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2/neu), members of the ErbB receptor tyrosine kinase family, are frequently overexpressed in breast cancer and are known to drive tumor growth and progression, making them promising targets for cancer therapy. Lapatinib is a selective competitive inhibitor of both the HER2 and EGFR tyrosine kinases. Although lapatinib showed significant activity in patients with HER2-positive breast cancer, the role of EGFR in the response of breast cancer to lapatinib has not been defined. Here, we examined the role of EGFR expression levels in the sensitivity of HER2-overexpressing breast cancer cells to lapatinib. Depletion of EGFR by EGFR small-interfering RNA knockdown did not affect lapatinib sensitivity in these cells, whereas treated HER2 siRNA knockdown cells became more resistant to lapatinib. We conclude that the in vitro activity of lapatinib is not dependent on EGFR expression level in HER2-overexpressing breast cancer cells.

A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation.

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.

Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activity.

Inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinases, such as erlotinib and gefitinib, have not been very effective in the treatment of breast cancer although many breast cancer cells express EGFR. To address this apparent paradox, we examined possible predictors of the sensitivity of 10 breast cancer cell lines to erlotinib in light of cyclin-dependent kinase 2 (CDK2), considered the farthest downstream kinase that controls cell cycling in the EGFR signaling pathway. Expression of EGFR and HER2 were not associated with sensitivity to erlotinib. Expression of phosphorylated (p-)tyrosine, p-Akt, phosphorylated extracellular signal-regulated kinase (p-ERK) 1/ERK2 (p42/p44), and p27 after treatment of erlotinib was not associated with erlotinib sensitivity. However, suppression of CDK2 activity after erlotinib treatment correlated with erlotinib sensitivity (P < 0.0001). Restoration of CDK2 activity partially restored proliferation and induced erlotinib resistance in erlotinib-sensitive cell lines, indicating that sensitivity to erlotinib in these breast cancer cells depends, at least in part, on CDK2 activity. p27, an inhibitor of CDK2, was not translocated into the nucleus in erlotinib-resistant cell lines. Knocking down p27 protein partially blocked erlotinib-induced cell death and cell cycle arrest. These findings indicate that the ability of erlotinib to suppress CDK2 activity is critical for cellular sensitivity to erlotinib, regardless of EGFR expression level, and that the presence of p27 in the cytoplasm also participates in erlotinib resistance.