Race-related Differences in Sipuleucel-T Response Among Men with Metastatic Castrate-Resistant Prostate Cancer.

PURPOSE: Sipuleucel-T is an autologous cellular immunotherapy that targets prostatic acid phosphatase (PAP) and is available for treatment of men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). In this single-arm, two-cohort, multicenter clinical study, potential racial differences in immune responses to sipuleucel-T in men with mCRPC were explored. PATIENTS AND METHODS: Patients' blood samples were obtained to assess serum cytokines, humoral responses, and cellular immunity markers pre- and post-treatment. Baseline cumulative product parameters (total nucleated and CD54+ cell counts, and CD54 upregulation) were evaluated. IgM titers against the immunogen PA2024, the target antigen PAP, prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) were quantified by ELISA. Cytotoxic T lymphocyte activity was determined by ELISpots, and cytokine and chemokine concentrations by Luminex. RESULTS: Twenty-nine African Americans (AA) and 28 non-African Americans (non-AA) with mCRPC received sipuleucel-T. Baseline total nucleated cell count, CD54+ cell count, CD54 expression, and cumulative product parameters were higher in non-AA. Although PSA baseline levels were higher in AA, there were no racial differences in IgM antibody and IFN- ELISpots responses against PA2024, PAP, PSA and PSMA pre- and post-treatment. Expression of co-stimulatory receptor ICOS on CD4+ and CD8+ T cells, and the levels of Th1 cytokine granulocyte-macrophage colony-stimulating factor and chemokines CCL4 and CCL5, were significantly higher in AA pre- and/or post-treatment. Despite no difference in the overall survival, PSA changes from baseline were significantly different between the two races. CONCLUSIONS: The data suggest that immune correlates in blood differ in AA and non-AA with mCRPC pre- and post-sipuleucel-T.

Multiplicity of benign breast disease lesions and breast cancer risk in African American women.

The risk of developing subsequent breast cancer is higher in women diagnosed with benign breast disease (BBD) but these studies were primarily performed in non-Hispanic white populations. Still, these estimates have been used to inform breast cancer risk models that are being used clinically across all racial and ethnic groups. Given the high breast cancer mortality rates among African American (AA) women, it is critical to study BBD in this population, to ensure the risk models that include this information perform adequately. This study utilized data from AA women who underwent benign breast biopsies at a hospital served by the University Pathology Group in Detroit, Michigan, from 1998 to 2010. Patients were followed for subsequent breast cancers through the population-based Metropolitan Detroit Cancer Surveillance System (MDCSS). BBD lesion scores were assigned to represent the severity or extent of benign breast lesions, with higher scores indicating a greater number of distinct lesion types. Of 3,461 eligible AA women with BBD in the cohort, 6.88% (n=238) subsequently developed breast cancer. Examined individually, six of the eleven lesions (apocrine metaplasia, ductal hyperplasia, lobular hyperplasia, intraductal papilloma, sclerosing adenosis, columnar alterations and radial scars) were significantly associated with increased risk of breast cancer after adjustment for age and year of biopsy and were further considered in multiple lesion models. For every different type of benign breast lesion, subsequent risk of breast cancer increased by 25% (RR=1.25, 95% CI: 1.10, 1.42) after adjustment for age at biopsy and proliferative versus non-proliferative disease. In summary, this study affirms the increased breast cancer risk in AA women with BBD, particularly in those with multiple lesions. These findings have implications for the management of breast cancer risk in millions of women affected by BBD, a high risk group that could benefit from personalized surveillance and risk reduction strategies.

18 F-sodium fluoride positron emission tomography quantitation of bone metastases in African American and non-African American men with metastatic prostate cancer.

BACKGROUND: Bone is the most common site of metastases in men with prostate cancer. The objective of this study was to explore potential racial differences in the distribution of tumor metastases in the axial and appendicular skeleton. METHODS: We conducted a retrospective review of patients with metastatic prostate cancer to the bone as detected by 18 F-sodium fluoride positron emission tomography/computed tomography (18 F-NaF PET/CT) scans. In addition to describing patients' demographics and clinical characteristics, the metastatic bone lesions, and healthy bone regions were detected and quantified volumetrically using a quantitative imaging platform (TRAQinform IQ, AIQ Solutions). RESULTS: Forty men met the inclusion criteria with 17 (42%) identifying as African Americans and 23 (58%) identifying as non-African Americans. Most of the patients had axial (skull, ribcage, and spine) disease. The location and the number of lesions in the skeleton of metastatic prostate cancer patients with low disease burden were not different by race. CONCLUSIONS: In low-disease burden patients with metastatic prostate cancer, there were no overall differences by race in the location and number of lesions in axial or appendicular skeleton. Therefore, given equal access to molecular imaging, African Americans might derive similar benefits. Whether this holds true for patients with a higher disease burden or for other molecular imaging techniques is a topic for further study.

Sprouty4 negatively regulates ERK/MAPK signaling and the transition from in situ to invasive breast ductal carcinoma.

Breast ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive ductal carcinoma (IDC). It is still unclear which DCIS will become invasive and which will remain indolent. Patients often receive surgery and radiotherapy, but this early intervention has not produced substantial decreases in late-stage disease. Sprouty proteins are important regulators of ERK/MAPK signaling and have been studied in various cancers. We hypothesized that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling and that its loss/reduced expression is a mechanism by which DCIS lesions progress toward IDC, including triple-negative disease. Using immunohistochemistry, we found reduced Sprouty4 expression in IDC patient samples compared to DCIS, and that ERK/MAPK phosphorylation had an inverse relationship to Sprouty4 expression. These observations were reproduced using a 3D culture model of disease progression. Knockdown of Sprouty4 in MCF10.DCIS cells increased ERK/MAPK phosphorylation as well as their invasive capability, while overexpression of Sprouty4 in MCF10.CA1d IDC cells reduced ERK/MAPK phosphorylation, invasion, and the aggressive phenotype exhibited by these cells. Immunofluorescence experiments revealed reorganization of the actin cytoskeleton and relocation of E-cadherin back to the cell surface, consistent with the restoration of adherens junctions. To determine whether these effects were due to changes in ERK/MAPK signaling, MEK1/2 was pharmacologically inhibited in IDC cells. Nanomolar concentrations of MEK162/binimetinib restored an epithelial-like phenotype and reduced pericellular proteolysis, similar to Sprouty4 overexpression. From these data we conclude that Sprouty4 acts to control ERK/MAPK signaling in DCIS, thus limiting the progression of these premalignant breast lesions.

Inflammation markers on benign breast biopsy are associated with risk of invasive breast cancer in African American women.

PURPOSE: Markers of inflammation, including crown-like structures of the breast (CLS-B) and infiltrating lymphocytes (IL), have been identified in breast tissue and associated with increased risk of breast cancer (BrCa), however most of this work has been performed in primarily non-Hispanic white women. Here, we examined whether CLS-B and IL are associated with invasive BrCa in African American (AA) women. METHODS: We assessed breast biopsies from three 5-year age-matched groups: BrCa-free AA women (50 Volunteer) from the Komen Normal Tissue Bank (KTB) and AA women with a clinically-indicated biopsy diagnosed with benign breast disease (BBD) from our Detroit cohort who developed BrCa (55 BBD-cancer) or did not develop BrCa (47 BBD only, year of biopsy matched to BBD-cancer). Mean adipocyte diameter and total adipose area were estimated from digital images using the Adiposoft plugin from ImageJ. Associations between CLS-B, IL, and BrCa among KTB and Detroit biopsies were assessed using multivariable multinomial and conditional logistic regression models. RESULTS: Among all biopsies, Volunteer and BBD only biopsies did not harbor CLS-B or IL at significantly different rates after adjusting for logarithm of adipocyte area, adipocyte diameter, and BMI. Among clinically-indicated BBD biopsies, BBD-cancer biopsies were more likely to exhibit CLS-B (odds ratio (OR) = 3.36, 95% Confidence Interval (CI): 1.33-8.48) or IL (OR = 4.95, 95% CI 1.76-13.9) than BBD only biopsies after adjusting for total adipocyte area, adipocyte diameter, proliferative disease, and BMI. CONCLUSIONS: CLS-B and IL may serve as histological markers of BrCa risk in benign breast biopsies from AA women.

Heritable Susceptibility to Breast Cancer among African-American Women in the Detroit Research on Cancer Survivors Study.

BACKGROUND: African-American women have high rates of breast cancer associated with hereditary features. However, no studies have reported the prevalence of inherited variation across all genes known to be breast cancer risk factors among African-American patients with breast cancer not selected for high-risk characteristics. METHODS: We evaluated 182 African-American women diagnosed with invasive breast cancer in metropolitan Detroit via targeted capture and multiplex sequencing of 13 well-established breast cancer risk genes and five suggested breast cancer risk genes. RESULTS: We identified 24 pathogenic variants in 23 women [12.6%; 95% confidence interval (CI), 8.2%-18.4%] and five genes (BRCA2, BRCA1, ATM, RAD50, CDH1). BRCA1 and BRCA2 accounted for 58.3% of all pathogenic variants. An additional six pathogenic variants were found in suggested breast cancer risk genes (MSH6, MUTYH, NF1, BRIP1). CONCLUSIONS: The prevalence of germline pathogenic variants is relatively high among African-American patients with breast cancer unselected for high-risk characteristics across a broad spectrum of genes. IMPACT: This study helps to define the genomic landscape of breast cancer susceptibility in African-American women who could benefit from enhanced surveillance and screening.

Role of TET1 and 5hmC in an Obesity-Linked Pathway Driving Cancer Stem Cells in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of estrogen receptor, progesterone receptor, and the HER2 but is enriched with cancer stem cell-like cells (CSC). CSCs are the fraction of cancer cells recognized as the source of primary malignant tumors that also give rise to metastatic recurrence. 5-Hydroxymethylcytosine (5hmC) is a DNA epigenetic feature derived from 5-methylcytosine by action of tet methylcytosine dioxygenase enzymes (e.g., TET1); and although TET1 and 5hmC are required to maintain embryonic stem cells, the mechanism and role in CSCs remain unknown. Data presented in this report support the conclusion that TET1 and TET1-dependent 5hmC mediate hydrogen peroxide (H2O2)-dependent activation of a novel gene expression cascade driving self-renewal and expansion of CSCs in TNBC. Evidence presented also supports that the H2O2 affecting this pathway arises due to endogenous mechanisms-including downregulation of antioxidant enzyme catalase in TNBC cells-and by exogenous routes, such as systemic inflammation and oxidative stress coupled with obesity, a known risk factor for TNBC incidence and recurrence. IMPLICATIONS: This study elucidates a pathway dependent on H2O2 and linked to obesity-driven TNBC tumor-initiating CSCs; thus, it provides new understanding that may advance TNBC prevention and treatment strategies.

Cetuximab PET delineated changes in cellular distribution of EGFR upon dasatinib treatment in triple negative breast cancer.

BACKGROUND: At least 50% of triple negative breast cancer (TNBC) overexpress the epidermal growth factor receptor, EGFR, which paved the way for clinical trials investigating its blockade. Outcomes remained dismal stemming from mechanisms of resistance particularly the nuclear cycling of EGFR, which is enhanced by Src activation. Attenuation of Src reversed nuclear translocation, restoring EGFR to the cell surface. Herein, we hypothesize that changes in cellular distribution of EGFR upon Src inhibition with dasatinib can be annotated through the EGFR immunopositron emission tomography (immunoPET) radiotracer, [89Zr]Zr-cetuximab. METHODS: Nuclear and non-nuclear EGFR levels of dasatinib-treated vs. untreated MDA-MB-231 and MDA-MB-468 cells were analyzed via immunoblots. Both treated and untreated cells were exposed to [89Zr]Zr-cetuximab to assess binding at 4 °C and 37 °C. EGFR-positive MDA-MB-231, MDA-MB-468, and a patient-derived xenograft were treated with dasatinib or vehicle followed by cetuximab PET imaging to compare EGFR levels. After imaging, the treated mice were separated into two groups: one cohort continued with dasatinib with the addition of cetuximab while the other cohort received dasatinib alone. Correlations between the radiotracer uptake vs. changes in tumor growth and EGFR expression from immunoblots were analyzed. RESULTS: Treated cells displayed higher binding of [89Zr]Zr-cetuximab to the cell membrane at 4 °C and with greater internalized activity at 37 °C vs. untreated cells. In all tumor models, higher accumulation of the radiotracer in dasatinib-treated groups was observed compared to untreated tumors. Treated tumors displayed significantly decreased pSrc (Y416) with retained total Src levels compared to control. In MDA-MB-468 and PDX tumors, the analysis of cetuximab PET vs. changes in tumor volume showed an inverse relationship where high tracer uptake in the tumor demonstrated minimal tumor volume progression. Furthermore, combined cetuximab and dasatinib treatment showed better tumor regression compared to control and dasatinib-only-treated groups. No benefit was achieved in MDA-MB-231 xenografts with the addition of cetuximab, likely due to its KRAS-mutated status. CONCLUSIONS: Cetuximab PET can monitor effects of dasatinib on EGFR cellular distribution and potentially inform treatment response in wild-type KRAS TNBC.

CLCA2 expression is associated with survival among African American women with triple negative breast cancer.

PURPOSE: Black/African American (AA) women are twice as likely to be diagnosed with triple negative breast cancer (TNBC) compared to whites, an aggressive breast cancer subtype associated with poor prognosis. There are no routinely used targeted clinical therapies for TNBC; thus there is a clear need to identify prognostic markers and potential therapeutic targets. METHODS: We evaluated expression of 27,016 genes in 155 treatment-naïve TN tumors from AA women in Detroit. Associations with survival were evaluated using Cox proportional hazards models adjusting for stage and age at diagnosis, and p-values were corrected using a false discovery rate. Our validation sample consisted of 494 TN tumors using four publically available data sets. Meta-analyses were performed using summary statistics from the four validation results. RESULTS: In the Detroit AA cohort, CLCA2 [Hazard ratio (HR) = 1.56, 95% confidence interval (CI) 1.31-1.86, nominal p = 5.1x10-7, FDR p = 0.014], SPIC [HR = 1.47, 95%CI 1.26-1.73, nominal p = 1.8x10-6, FDR p = 0.022], and MIR4311 [HR = 1.57, 95% CI 1.31-1.92, nominal p = 2.5x10-5, FDR p = 0.022] expression were associated with overall survival. Further adjustment for treatment and breast cancer specific survival analysis did not substantially alter effect estimates. CLCA2 was also associated with increased risk of death in the validation cohorts [HR = 1.14, 95% CI 1.05-1.24, p = 0.038, p-heterogeneity = 0.88]. CONCLUSIONS: We identified CLCA2 as a potential prognostic marker for TNBC in AA women.

Downregulation of Rap1Gap: A Switch from DCIS to Invasive Breast Carcinoma via ERK/MAPK Activation.

Diagnosis of breast ductal carcinoma in situ (DCIS) presents a challenge since we cannot yet distinguish those cases that would remain indolent and not require aggressive treatment from cases that may progress to invasive ductal cancer (IDC). The purpose of this study is to determine the role of Rap1Gap, a GTPase activating protein, in the progression from DCIS to IDC. Immunohistochemistry (IHC) analysis of samples from breast cancer patients shows an increase in Rap1Gap expression in DCIS compared to normal breast tissue and IDCs. In order to study the mechanisms of malignant progression, we employed an in vitro three-dimensional (3D) model that more accurately recapitulates both structural and functional cues of breast tissue. Immunoblotting results show that Rap1Gap levels in MCF10.Ca1D cells (a model of invasive carcinoma) are reduced compared to those in MCF10.DCIS (a model of DCIS). Retroviral silencing of Rap1Gap in MCF10.DCIS cells activated extracellular regulated kinase (ERK) mitogen-activated protein kinase (MAPK), induced extensive cytoskeletal reorganization and acquisition of mesenchymal phenotype, and enhanced invasion. Enforced reexpression of Rap1Gap in MCF10.DCIS-Rap1GapshRNA cells reduced Rap1 activity and reversed the mesenchymal phenotype. Similarly, introduction of dominant negative Rap1A mutant (Rap1A-N17) in DCIS-Rap1Gap shRNA cells caused a reversion to nonmalignant phenotype. Conversely, expression of constitutively active Rap1A mutant (Rap1A-V12) in noninvasive MCF10.DCIS cells led to phenotypic changes that were reminiscent of Rap1Gap knockdown. Thus, reduction of Rap1Gap in DCIS is a potential switch for progression to an invasive phenotype. The Graphical Abstract summarizes these findings.

Treating triple negative breast cancer cells with erlotinib plus a select antioxidant overcomes drug resistance by targeting cancer cell heterogeneity.

Among breast cancer patients, those diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst prog-nosis. TNBC does not express estrogen receptor-alpha, progesterone receptor, or the HER2 oncogene; therefore, TNBC lacks targets for molecularly-guided therapies. The concept that EGFR oncogene inhibitor drugs could be used as targeted treatment against TNBC has been put forth based on estimates that 30-60% of TNBC express high levels of EGFR. However, results from clinical trials testing EGFR inhibitors, alone or in combination with cytotoxic chemotherapy, did not improve patient outcomes. Results herein offer an explanation as to why EGFR inhibitors failed TNBC patients and support how combining a select antioxidant and an EGFR-specific small molecule kinase inhibitor (SMKI) could be an effective, novel therapeutic strategy. Treatment with CAT-SKL-a re-engineered protein form of the antioxidant enzyme catalase-inhibited cancer stem-like cells (CSCs), and treatment with the EGFR-specific SMKI erlotinib inhibited non-CSCs. Thus, combining the antioxidant CAT-SKL with erlotinib targeted both CSCs and bulk cancer cells in cultures of EGFR-expressing TNBC-derived cells. We also report evidence that the mechanism for CAT-SKL inhibition of CSCs may depend on antioxidant-induced downregulation of a short alternative mRNA splicing variant of the methyl-CpG binding domain 2 gene, isoform MBD2c.

Targeting MET and EGFR crosstalk signaling in triple-negative breast cancers.

There is a vital need for improved therapeutic strategies that are effective in both primary and metastatic triple-negative breast cancer (TNBC). Current treatment options for TNBC patients are restricted to chemotherapy; however tyrosine kinases are promising druggable targets due to their high expression in multiple TNBC subtypes. Since coexpression of receptor tyrosine kinases (RTKs) can promote signaling crosstalk and cell survival in the presence of kinase inhibitors, it is likely that multiple RTKs will need to be inhibited to enhance therapeutic benefit and prevent resistance. The MET and EGFR receptors are actionable targets due to their high expression in TNBC; however crosstalk between MET and EGFR has been implicated in therapeutic resistance to single agent use of MET or EGFR inhibitors in several cancer types. Therefore it is likely that dual inhibition of MET and EGFR is required to prevent crosstalk signaling and acquired resistance. In this study, we evaluated the heterogeneity of MET and EGFR expression and activation in primary and metastatic TNBC tumorgrafts and determined the efficacy of MET (MGCD265 or crizotinib) and/or EGFR (erlotinib) inhibition against TNBC progression. Here we demonstrate that combined MET and EGFR inhibition with either MGCD265 and erlotinib treatment or crizotinib and erlotinib treatment were highly effective at abrogating tumor growth and significantly decreased the variability in treatment response compared to monotherapy. These results advance our understanding of the RTK signaling architecture in TNBC and demonstrate that combined MET and EGFR inhibition may be a promising therapeutic strategy for TNBC patients.

Phase I Safety, Pharmacokinetic, and Pharmacodynamic Study of the Poly(ADP-ribose) Polymerase (PARP) Inhibitor Veliparib (ABT-888) in Combination with Irinotecan in Patients with Advanced Solid Tumors.

PURPOSE: PARP is essential for recognition and repair of DNA damage. In preclinical models, PARP inhibitors modulate topoisomerase I inhibitor-mediated DNA damage. This phase I study determined the MTD, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib, an orally bioavailable PARP1/2 inhibitor, in combination with irinotecan. EXPERIMENTAL DESIGN: Patients with advanced solid tumors were treated with 100 mg/m(2) irinotecan on days 1 and 8 of a 21-day cycle. Twice-daily oral dosing of veliparib (10-50 mg) occurred on days 3 to 14 (cycle 1) and days -1 to 14 (subsequent cycles) followed by a 6-day rest. PK studies were conducted with both agents alone and in combination. Paired tumor biopsies were obtained after irinotecan alone and veliparib/irinotecan to evaluate PARP1/2 inhibition and explore DNA damage signals (nuclear γ-H2AX and pNBS1). RESULTS: Thirty-five patients were treated. DLTs included fatigue, diarrhea, febrile neutropenia, and neutropenia. The MTD was 100 mg/m(2) irinotecan (days 1 and 8) combined with veliparib 40 mg twice daily (days -1-14) on a 21-day cycle. Of 31 response-evaluable patients, there were six (19%) partial responses. Veliparib exhibited linear PK, and there were no apparent PK interactions between veliparib and irinotecan. At all dose levels, veliparib reduced tumor poly(ADP-ribose) (PAR) content in the presence of irinotecan. Several samples showed increases in γ-H2AX and pNBS1 after veliparib/irinotecan compared with irinotecan alone. CONCLUSIONS: Veliparib can be safely combined with irinotecan at doses that inhibit PARP catalytic activity. Preliminary antitumor activity justifies further evaluation of the combination. Clin Cancer Res; 22(13); 3227-37. ©2016 AACR.

Protein expression of DNA damage repair proteins dictates response to topoisomerase and PARP inhibitors in triple-negative breast cancer.

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.

Abrogating phosphorylation of eIF4B is required for EGFR and mTOR inhibitor synergy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive disease. They have a high rate of relapse and often develop resistance to standard chemotherapy. Many TNBCs have elevated epidermal growth factor receptor (EGFR) but are resistant to EGFR inhibitors as monotherapy. In this study, we sought to find a combination therapy that could sensitize TNBC to EGFR inhibitors. Phospho-mass spectrometry was performed on the TNBC cell line, BT20, treated with 0.5 µM gefitinib. Immunoblotting measured protein levels and phosphorylation. Colony formation and growth assays analyzed the treatment on cell proliferation, while MTT assays determined the synergistic effect of inhibitor combination. A Dual-Luciferase reporter gene plasmid measured translation. All statistical analysis was done on CalucuSyn and GraphPad Prism using ANOVAs. Phospho-proteomics identified the mTOR pathway to be of interest in EGFR inhibitor resistance. In our studies, combining gefitinib and temsirolimus decreased cell growth and survival in a synergistic manner. Our data identified eIF4B, as a potentially key fragile point in EGFR and mTOR inhibitor synergy. Decreased eIF4B phosphorylation correlated with drops in growth, viability, clonogenic survival, and cap-dependent translation. Taken together, these data suggest EGFR and mTOR inhibitors abrogate growth, viability, and survival via disruption of eIF4B phosphorylation leading to decreased translation in TNBC cell lines. Further, including an mTOR inhibitor along with an EGFR inhibitor in TNBC with increased EGFR expression should be further explored. Additionally, translational regulation may play an important role in regulating EGFR and mTOR inhibitor synergy and warrant further investigation.

EGFR Tyrosine 845 Phosphorylation-Dependent Proliferation and Transformation of Breast Cancer Cells Require Activation of p38 MAPK.

Phosphorylation of epidermal growth factor receptor (EGFR) on tyrosine 845 by c-Src has been shown to be important for cell proliferation and migration in several model systems. This cross talk between EGFR and Src family kinases (SFKs) is one mechanism for resistance to EGFR inhibitors both in cell models and in the clinic. Here, we show that phosphorylation of tyrosine 845 on EGFR is required for proliferation and transformation using several cell models of breast cancer. Overexpression of EGFR-Y845F or treating cells with the SFK inhibitor dasatinib abrogated tyrosine 845 phosphorylation, yet had little to no effect on other EGFR phosphorylation sites or EGFR kinase activity. Abrogation of Y845 phosphorylation inhibited cell proliferation and transformation, even though extracellular signal-regulated kinase (ERK) and Akt remained active under these conditions. Importantly, cotransfection of mitogen-activated protein kinase (MAPK) kinase 3 and p38 MAPK restored cell proliferation in the absence of EGFR tyrosine 845 phosphorylation. Taken together, these data demonstrate a novel role for p38 MAPK signaling downstream of EGFR tyrosine 845 phosphorylation in the regulation of breast cancer cell proliferation and transformation and implicate SFK inhibitors as a potential therapeutic mechanism for overcoming EGFR tyrosine kinase inhibitor resistance in breast cancer.

Genomic amplification and a role in drug-resistance for the KDM5A histone demethylase in breast cancer.

Lysine-specific demethylase 5A (KDM5A), an enzyme that removes activating H3K4 di- and trimethylation marks, plays critical roles in controlling transcription and chromatin architecture, yet its biological functions largely remain uncharacterized, particularly in the context of human cancer. In the present study, we found that the KDM5A gene was significantly amplified and over-expressed in various human tumors, including breast cancer. Reducing the expression of KDM5A by shRNA knockdown inhibited proliferation of KDM5A-amplified breast cancer cells. More importantly, we demonstrated that KDM5A over-expression was associated with breast cancer drug resistance. Furthermore, knockdown of KDM5A gene expression altered H3K4 methylation and induced upregulation of CDK inhibitors as well as genes mediating apoptotic cell death. Taken together, our study strongly links KDM5A histone demethylase activity to breast cancer proliferation and drug resistance, and suggests KDM5A is a potential target for breast cancer therapy.

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met.

INTRODUCTION: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC). METHODS: The SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student's t test was used for all statistical analysis. RESULTS: Here we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI. CONCLUSIONS: Taken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions.

A targeted enzyme approach to sensitization of tyrosine kinase inhibitor-resistant breast cancer cells.

Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of potential use in patients with breast cancer. Unfortunately, in clinical studies, gefitinib is often ineffective indicating that resistance to EGFR inhibitors may be a common occurrence in cancer of the breast. EGFR has been shown to be overexpressed in breast cancer, and in particular remains hyperphosphorylated in cell lines such as MDA-MB-468 that are resistant to EGFR inhibitors. Here, we investigate the cause of this sustained phosphorylation and the molecular basis for the ineffectiveness of gefitinib. We show that reactive oxygen species (ROS), known to damage cellular macromolecules and to modulate signaling cascades in a variety of human diseases including cancers, appear to play a critical role in mediating EGFR TKI-resistance. Furthermore, elimination of these ROS through use of a cell-penetrating catalase derivative sensitizes the cells to gefitinib. These results suggest a new approach for the treatment of TKI-resistant breast cancer patients specifically, the targeting of ROS and attendant downstream oxidative stress and their effects on signaling cascades.

Src family kinases mediate epidermal growth factor receptor signaling from lipid rafts in breast cancer cells.

Activation of the epidermal growth factor receptor (EGFR) regulates cellular proliferation, survival, and migration of breast cancer cells. In particular, EGFR recruits signaling proteins to the cell membrane leading to their phosphorylation and activation. However, EGFR also localizes to other cellular structures, including endosomes, mitochondrion, and nuclei. Recently, we demonstrated that lipid raft localization of EGFR in triple-negative breast cancer cell lines promotes EGFR protein-dependent, EGFR kinase-independent activation of Akt. Here, we further define the mechanism by which lipid rafts regulate EGFR signaling to Akt. Specifically, we show that the non-receptor tyrosine kinase c-Src co-localizes and co-associates with EGFR and lipid rafts. Breast cancer cells resistant to treatment with EGFR inhibitors, were also resistant to treatment with Src family kinase (SFK) inhibitors; however, the combination of EGFR and SFK inhibitors synergistically decreases cell viability. We found that this decrease in cell viability observed with EGFR and SFK inhibitor co-treatment correlates with loss of Akt phosphorylation. In addition, we found that in breast cancer cell lines with EGFR and c-Src co-localized to lipid rafts, phospho-inositide 3 kinase (PI3K) was also associated with lipid rafts. Together, the data herein suggest that lipid rafts provide a platform for the interaction of EGFR, c-Src, and PI3K, leading to activation of cellular survival signaling in breast cancer cells.