Evaluation of PD-L1, tumor-infiltrating lymphocytes, and CD8+ and FOXP3+ immune cells in HER2-positive breast cancer treated with neoadjuvant therapies.

BACKGROUND: The tumor immune microenvironment plays a critical role in the prognosis and outcome of breast cancers. This study examined the role of tumor-infiltrating lymphocytes (TILs), CD8+, FOXP3+ lymphocytes, PD-L1 expression, and other clinicopathological parameters in HER2+ breast cancer and correlate with tumor response to neoadjuvant therapy. METHODS: We included 173 HER2+ patients treated with neoadjuvant HER2-targeted chemotherapy regimens from 2010 to 2016. 67 cases had biopsy blocks to evaluate TIL, CD8, FOXP3, and PD-L1 immunohistochemistry staining. Tumors were classified as pCR vs non-pCR group. Clinicopathological parameters, TIL, CD8+ and FOXP3+ cell count, and PD-L1 expression were correlated with pCR rate. RESULTS: Univariate analyses showed that pCR rate was significantly correlated with low PR, low ER, high Ki-67, high FOXP3, HER2 IHC3+ , high HER2 ratio and copy number. By multivariate analysis, Ki-67 was the only variable significantly correlated with pCR. PD-L1 expression was detected in 9.2% cases. TIL hotspot has a non-significant correlation with pCR rate (p = 0.096). CONCLUSIONS: High Ki-67 is a strong predictor for pCR in HER2+ breast cancer. TIL and FOXP3 T cells may play a role in tumor response in HER2+ cancer. PD-L1 is expressed in a subset of HER2+ breast cancer, supporting a role of immunotherapy in treating a subset of HER2+ breast cancers. The role of PD-L1, TIL, and other markers of immunogenicity as predictors of response to neoadjuvant chemotherapy in HER2+ breast cancer should be further evaluated.

Evasion of anti-growth signaling: A key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds.

The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting.

Insulin-like growth factor-1 receptor signaling increases the invasive potential of human epidermal growth factor receptor 2-overexpressing breast cancer cells via Src-focal adhesion kinase and forkhead box protein M1.

Resistance to the human epidermal growth factor receptor (HER2)-targeted antibody trastuzumab is a major clinical concern in the treatment of HER2-positive metastatic breast cancer. Increased expression or signaling from the insulin-like growth factor-1 receptor (IGF-1R) has been reported to be associated with trastuzumab resistance. However, the specific molecular and biologic mechanisms through which IGF-1R promotes resistance or disease progression remain poorly defined. In this study, we found that the major biologic effect promoted by IGF-1R was invasion, which was mediated by both Src-focal adhesion kinase (FAK) signaling and Forkhead box protein M1 (FoxM1). Cotargeting IGF-1R and HER2 using either IGF-1R antibodies or IGF-1R short hairpin RNA in combination with trastuzumab resulted in significant but modest growth inhibition. Reduced invasion was the most significant biologic effect achieved by cotargeting IGF-1R and HER2 in trastuzumab-resistant cells. Constitutively active Src blocked the anti-invasive effect of IGF-1R/HER2 cotargeted therapy. Furthermore, knockdown of FoxM1 blocked IGF-1-mediated invasion, and dual targeting of IGF-1R and HER2 reduced expression of FoxM1. Re-expression of FoxM1 restored the invasive potential of IGF-1R knockdown cells treated with trastuzumab. Overall, our results strongly indicate that therapeutic combinations that cotarget IGF-1R and HER2 may reduce the invasive potential of cancer cells that are resistant to trastuzumab through mechanisms that depend in part on Src and FoxM1.

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.

Systemic therapy for early-stage HER2-positive breast cancers: time for a less-is-more approach?

Trastuzumab-based chemotherapy has dramatically improved outcomes for patients with all stages of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Additional HER2-directed agents that have recently been approved are also expected to improve outcomes. Patients with small, lymph node-negative, HER2-positive breast cancers who are treated with trastuzumab-based chemotherapy demonstrate especially favorable responses, with 5-year recurrence rates of <5%. In this review, recent data regarding response rates among patients with early-stage HER2-positive breast cancer treated with trastuzumab-based chemotherapy are discussed. This review supports future studies of the possible omission of chemotherapy in a subset of patients with HER2-positive cancers, specifically those that coexpress hormone receptors.

Therapeutic implications of estrogen receptor signaling in HER2-positive breast cancers.

There is considerable pre-clinical and clinical evidence demonstrating that HER2-positive breast cancers that express estrogen receptor (ER) exhibit intrinsic resistance to endocrine therapy. Therefore, in general, chemotherapy in combination with HER2-directed agents is recommended for all but the smallest HER2-positive early stage breast cancers regardless of ER status. This paradigm has recently come into question when responses to neo-adjuvant HER2-directed regimens were noted to vary based on ER expression, and pathologic complete response was noted not to be prognostic for ER-positive, HER2-positive breast cancers. These and other data suggest the possibility that a subset of HER2-positive, ER-positive breast cancers are driven primarily by ER, and biologically behave more like HER2-negative, ER-positive breast cancers. Identification of this subset of HER2-positive breast cancers is essential to avoid over-treatment of patients with small HER2-positive, ER-positive breast cancers, who may be optimally treated with endocrine therapy alone, or in combination with a HER2-directed agent, thereby avoiding the use of chemotherapy. Crosstalk between the ER and HER2 pathways has been established as playing a role in both intrinsic and acquired resistance to endocrine agents. Emerging data suggests that crosstalk between these pathways is also involved in resistance to HER2-directed agents. Unraveling the role of the ER pathway in resistance to HER2-directed agents could potentially result in therapeutic approaches that can improve outcome for patients with ER-positive, HER2-positive breast cancer.

Using Online Cancer Genomics Databases to Provide Teaching Resources for Pharmacy Education.

Connecting scientific concepts with clinical applications is an important objective of pharmacy education. As the field of precision oncology expands, it is critical for pharmacy students to understand how genetic information informs cancer treatment decisions. However, to effectively teach students about pharmacogenomics and pharmacogenetics, faculty require relevant educational resources, including those that support higher-order learning. In this Commentary, we demonstrate the potential utility of publicly accessible cancer genomics databases as teaching resources for pharmacogenomics and pharmacogenetics in oncology pharmacy education. Using clinical data retrieved from a genomics database, we illustrate how case studies can be developed to target core competencies, including understanding tumor genomics profiling, somatic mutations and pharmacotherapy selection, and clinical pharmacogenetics testing. Cancer genomics databases provide readily available, cost-effective, clinical data resources that support active learning related to pharmacogenomics and pharmacogenetics education in oncology pharmacy curricula.

Phosphatase magnesium-dependent 1 δ (PPM1D), serine/threonine protein phosphatase and novel pharmacological target in cancer.

Aberrations in DNA damage response genes are recognized mediators of tumorigenesis and resistance to chemo- and radiotherapy. While protein phosphatase magnesium-dependent 1 δ (PPM1D), located on the long arm of chromosome 17 at 17q22-23, is a key regulator of cellular responses to DNA damage, amplification, overexpression, or mutation of this gene is important in a wide range of pathologic processes. In this review, we describe the physiologic function of PPM1D, as well as its role in diverse processes, including fertility, development, stemness, immunity, tumorigenesis, and treatment responsiveness. We highlight both the advances and limitations of current approaches to targeting malignant processes mediated by pathogenic alterations in PPM1D with the goal of providing rationale for continued research and development of clinically viable treatment approaches for PPM1D-associated diseases.

Correction: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling.

[This corrects the article DOI: 10.18632/oncotarget.21765.].

Clinicopathologic Factors Associated With Response to Neoadjuvant Anti-HER2-Directed Chemotherapy in HER2-Positive Breast Cancer.

BACKGROUND: HER2-targeted neoadjuvant therapy has high efficacy in treating HER2-positive breast cancer. Response to neoadjuvant therapy helps clinicians make treatment decisions and make estimates about prognosis. This study examined clinicopathologic features to determine which may be most predictive of response to neoadjuvant therapy in HER2+ breast cancer. PATIENTS AND METHODS: Patients with HER2+ breast cancer (n = 173) who had an initial biopsy performed between 2010 and 2016 were identified at our institution. Tumor response was evaluated on excisional specimens using the MD Anderson residual cancer burden (RCB) classification. Tumors with pathologic complete response (defined as no residual invasive carcinoma in the breast and lymph nodes) and RCB-I were classified as having response and tumors with RCB-II and -III as having no response. Patient age, tumor size, nuclear grade (1/2 vs. 3), mitosis, Nottingham grade, HER2 immunohistochemistry (1/2+ vs. 3+), HER2/CEP17 (chromosome enumeration probe 17) ratio, HER2 copy number, estrogen receptor, progesterone receptor, Ki-67, and tumor-infiltrating lymphocytes (TIL) were evaluated and correlated with response. TILs were evaluated for an average and also for the hot spot/total tumor stromal ratio. RESULTS: Small tumor size, low estrogen receptor and progesterone receptor expression, HER2 immunohistochemistry 3+, high Ki-67, high HER2/CEP17 ratio, and high HER2 copy number were significantly associated with response (all P < .05). TIL hot spot was associated with RCB in univariate (P < .05) but not multivariate analyses. CONCLUSION: Clinicopathologic features may help predict HER2+ breast cancer response to neoadjuvant therapy. Larger studies would be useful to confirm these associations, which may have relevance to clinical practice.

A novel unidirectional cross-talk from the insulin-like growth factor-I receptor to leptin receptor in human breast cancer cells.

Obesity is a major risk factor for the development and progression of breast cancer. Increased circulating levels of the obesity-associated hormones leptin and insulin-like growth factor-I (IGF-I) and overexpression of the leptin receptor (Ob-R) and IGF-I receptor (IGF-IR) have been detected in a majority of breast cancer cases and during obesity. Due to correlations between increased leptin, Ob-R, IGF-I, and IGF-IR in breast cancer, we hypothesized that molecular interactions may exist between these two signaling pathways. Coimmunoprecipitation and immunoblotting showed that IGF-IR and Ob-R interact in the breast cancer cell lines MDA-MB-231, MCF7, BT474, and SKBR3. Stimulation of cells with IGF-I promoted Ob-R phosphorylation, which was blocked by IGF-IR kinase inhibition. In addition, IGF-I activated downstream signaling molecules in the leptin receptor and IGF-IR pathways. In contrast to IGF-I, leptin did not induce phosphorylation of IGF-IR, indicating that receptor cross-signaling is unidirectional, occurring from IGF-IR to Ob-R. Our results show, for the first time, a novel interaction and cross-talk between the IGF-I and leptin receptors in human breast cancer cells.

Nordihydroguaiaretic acid, a cytotoxic insulin-like growth factor-I receptor/HER2 inhibitor in trastuzumab-resistant breast cancer.

The majority of patients with HER2-overexpressing metastatic breast cancer who initially respond to the HER2-targeted antibody trastuzumab show disease progression within 1 year. The identification of novel agents that effectively inhibit survival of cancer cells that have progressed on trastuzumab is critical for improving outcome for this patient population. In the current study, we show that the phenolic compound nordihydroguaiaretic acid (NDGA) promoted cell death of trastuzumab-naive and trastuzumab-refractory HER2-overexpressing breast cancer cells. NDGA induced DNA fragmentation, cleavage of poly(ADP-ribose) polymerase and caspase-3, and inhibition of colony formation. In addition, NDGA inhibited insulin-like growth factor-I and HER2 signaling in trastuzumab-refractory cells, with reduced downstream phosphatidylinositol-3 kinase/Akt signaling. Importantly, combination treatment with NDGA and trastuzumab suppressed proliferation and survival of trastuzumab-refractory cells to a greater degree than either agent alone, suggesting that NDGA increases the sensitivity of refractory cells to trastuzumab. Derivatives of NDGA are currently in clinical trial for other solid tumors. Our data strongly support further study of NDGA as a potential therapeutic against breast cancers that have progressed on trastuzumab.

Inhibition of mutant PPM1D enhances DNA damage response and growth suppressive effects of ionizing radiation in diffuse intrinsic pontine glioma.

BACKGROUND: Children with diffuse intrinsic pontine glioma (DIPG) succumb to disease within 2 years of diagnosis despite treatment with ionizing radiation (IR) and/or chemotherapy. Our aim was to determine the role of protein phosphatase, magnesium-dependent 1, delta (PPM1D) mutation, present in up to 25% of cases, in DIPG pathogenesis and treatment. METHODS: Using genetic and pharmacologic approaches, we assayed effects of PPM1D mutation on DIPG growth and murine survival. We assayed effects of targeting mutated PPM1D alone or with IR on signaling, cell cycle, proliferation, and apoptosis in patient-derived DIPG cells in vitro, in organotypic brain slices, and in vivo. RESULTS: PPM1D-mutated DIPG cell lines exhibited increased proliferation in vitro and in vivo, conferring reduced survival in orthotopically xenografted mice, through stabilization of truncated PPM1D protein and inactivation of DNA damage response (DDR) effectors p53 and H2A.X. PPM1D knockdown or treatment with PPM1D inhibitors suppressed growth of PPM1D-mutated DIPGs in vitro. Orthotopic xenografting of PPM1D short hairpin RNA-transduced or PPM1D inhibitor-treated, PPM1D-mutated DIPG cells into immunodeficient mice resulted in reduced tumor proliferation, increased apoptosis, and extended mouse survival. PPM1D inhibition had similar effects to IR alone on DIPG growth inhibition and augmented the anti-proliferative and pro-apoptotic effects of IR in PPM1D-mutated DIPG models. CONCLUSIONS: PPM1D mutations inactivate DDR and promote DIPG growth. Treatment with PPM1D inhibitors activated DDR pathways and enhanced the anti-proliferative and pro-apoptotic effects of IR in DIPG models. Our results support continued development of PPM1D inhibitors for phase I/II trials in children with DIPG.

Her2 cross talk and therapeutic resistance in breast cancer.

The HER2 receptor tyrosine kinase is amplified and/or overexpressed in approximately 30 percent of metastatic breast cancers. Interactions and cross signaling from the HER2 receptor to other growth factor receptors may potentially contribute to therapeutic resistance. In this review, we discuss HER2 receptor cross talk with the estrogen receptor and implications toward resistance to endocrine therapies. We also review mechanisms of resistance to the HER2-targeted antibody trastuzumab, including signaling from other members of the HER family, increased signaling through the PI3-kinase pathway, and cross talk from the insulin-like growth factor-I receptor to HER2. Finally, we will provide perspective on how HER2 receptor cross talk may provide critical information for developing novel therapeutic options for HER2-overexpressing breast cancers.

Lapatinib induces apoptosis in trastuzumab-resistant breast cancer cells: effects on insulin-like growth factor I signaling.

The majority of breast cancer patients who achieve an initial therapeutic response to the HER2-targeted antibody trastuzumab will show disease progression within 1 year. Thus, the identification of novel agents that effectively inhibit survival of cancer cells that have progressed on trastuzumab is critical. In the current study, we show that the dual epidermal growth factor receptor (EGFR)/human EGFR-2 (HER2) kinase inhibitor lapatinib induces apoptosis in trastuzumab-resistant cells derived from the HER2-overexpressing SKBR3 breast cancer line. Lapatinib inhibited EGFR and HER2 signaling in resistant cells, blocking activation of downstream Akt, mitogen-activated protein kinase [corrected] Importantly, lapatinib also inhibited insulin-like growth factor I (IGF-I) signaling and growth-promoting effects in parental and resistant cells, and the cytotoxic effects of lapatinib were further enhanced by the IGF-I receptor-blocking antibody alphaIR3. As increased IGF-I receptor signaling has been implicated in trastuzumab resistance, our data strongly support further study of lapatinib as a potential therapeutic in breast cancers that have progressed on trastuzumab.

Adenovirus type 5 E1A gene therapy for ovarian clear cell carcinoma: a potential treatment strategy.

Resistance of ovarian clear cell carcinoma (CCC) to platinum-based chemotherapy is associated with poor prognosis, and an effective treatment for advanced disease is urgently needed. HER2/neu is up-regulated more often in CCC than in other histologic types of epithelial ovarian cancer. The purpose of this study was to assess possible treatment for ovarian CCC with the anti-HER2 antibody trastuzumab or human adenovirus type 5 E1A. We treated 10 CCC cell lines with trastuzumab or E1A and assessed cell viability, proliferation, and colony formation and the expression of HER2 and wild-type p53 proteins and molecules downstream of those signaling pathways. HER2 protein was detected at various levels in all 10 cell lines by Western blotting and in 5 CCC cell lines by immunohistochemical staining; HER2 gene amplification was detected (by fluorescence in situ hybridization) in only one cell line (RMG-I). Trastuzumab did not inhibit proliferation in any of the four CCC cell lines tested (RMG-I, SKOV-2, OVTOKO, and OVSAYO). However, transfection with E1A (as compared with control vectors) reduced colony formation in all 10 CCC cell lines regardless of HER2 expression level. Infection of RMG-I and SMOV-2 cells with an adenoviral vector encoding E1A led to significant (P < 0.05) suppression of proliferation and enhancement of cell death; this effect required stabilization of p53 (but not p73) protein and was associated with the up-regulation of Bax and the cleavage of caspase-9. Other mechanisms, such as p53-independent apoptosis, may also be involved in E1A-mediated cell death in CCC. Finally, treatment with E1A prolonged survival in a CCC xenograft model (P < 0.001). E1A gene therapy, because of its ability to stabilize wild-type p53, is worth exploring as a treatment modality for women with ovarian CCC, which typically expresses wild-type p53.

ABP 980: promising trastuzumab biosimilar for HER2-positive breast cancer.

INTRODUCTION: Approval of the HER2-targeted antibody trastuzumab dramatically improved outcomes for patients with HER2-positive breast cancer. Multiple trastuzumab biosimilars, including ABP 980, are in clinical development. Biosimilars are not identical to the reference biologic, but exhibit equivalence and safety in analytical and clinical studies. Areas covered: A brief introduction to trastuzumab, overview of trastuzumab biosimilars, and detailed review of ABP 980 preclinical and clinical studies are included. We searched PubMed and 2016-2017 ASCO and ESMO conference proceedings for 'ABP 980' or 'trastuzumab biosimilar'. 'ABP 980 and breast cancer' or 'trastuzumab biosimilar and breast cancer' were used to search clinicaltrials.gov for phase III trials. Analytical studies of ABP 980 pharmacokinetics (PK) or pharmacodynamics (PD), phase I studies of ABP 980 safety and PK/PD, and phase III studies of clinical efficacy vs trastuzumab are included. Expert opinion: Questions remain regarding long-term impact of biosimilars on overall healthcare costs, insurance coverage of multiple approved biosimilars, and extensive clinical safety and efficacy follow-up. By producing a competitive market, trastuzumab biosimilars are anticipated to improve access to standard of care therapies, although real-world evidence remains to be obtained. Increased global access to HER2-targeted therapy may eventually alter the landscape of breast cancer and survival rates.

Targeting forkhead box M1 transcription factor in breast cancer.

Breast cancer continues to be the most commonly diagnosed malignancy and second most common cause of cancer-related deaths among women in the United States. Improved understanding of the molecular heterogeneity of breast tumors and the approval of multiple targeted therapies have revolutionized the treatment landscape and long-term survival rates for patients with breast cancer. Despite the development of highly effective targeted agents, drug resistance and disease progression remain major clinical concerns. Improved understanding of the molecular mechanisms mediating drug resistance will allow new treatments to be developed. The forkhead box M1 (FoxM1) transcription factor is overexpressed in breast cancer and strongly associated with resistance to targeted therapies and chemotherapy. FoxM1 regulates all hallmarks of cancer, including proliferation, mitosis, EMT, invasion, and metastasis. Inhibition of FoxM1 transcription factor function is a potential strategy for overcoming breast cancer progression. In this research update, we review the role of FoxM1 in breast cancer and pharmacological approaches for blocking FoxM1 transcription factor function. Future preclinical studies should evaluate combination drug strategies to inhibit FoxM1 function and upstream kinase signaling pathways as potential strategies to treat resistant and metastatic breast cancers.

Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells.

The majority of breast cancer patients who achieve an initial therapeutic response to the human epidermal growth factor receptor 2 (HER-2)-targeted antibody trastuzumab will show disease progression within 1 year. We previously reported the characterization of SKBR3-derived trastuzumab-resistant pools. In the current study, we show that HER-2 interacts with insulin-like growth factor-I receptor (IGF-IR) uniquely in these resistant cells and not in the parental trastuzumab-sensitive cells. The occurrence of cross talk between IGF-IR and HER-2 exclusively in resistant cells is evidenced by the IGF-I stimulation resulting in increased phosphorylation of HER-2 in resistant cells, but not in parental cells, and by the inhibition of IGF-IR tyrosine kinase activity leading to decreased HER-2 phosphorylation only in resistant cells. In addition, inhibition of IGF-IR tyrosine kinase activity by I-OMe-AG538 increased sensitivity of resistant cells to trastuzumab. HER-2/IGF-IR interaction was disrupted on exposure of resistant cells to the anti-IGF-IR antibody alpha-IR3 and, to a lesser extent, when exposed to the anti-HER-2 antibody pertuzumab. Heterodimer disruption by alpha-IR3 dramatically restored sensitivity to trastuzumab and resistant cells showed a slightly increased sensitivity to pertuzumab versus parental cells. Neither alpha-IR3 nor pertuzumab decreased HER-2 phosphorylation, suggesting that additional sources of phosphorylation other than IGF-IR exist when HER-2 and IGF-IR are not physically bound. Our data support a unique interaction between HER-2 and IGF-IR in trastuzumab-resistant cells such that cross talk occurs between IGF-IR and HER-2. These data suggest that the IGF-IR/HER-2 heterodimer contributes to trastuzumab resistance and justify the need for further studies examining this complex as a potential therapeutic target in breast cancers that have progressed while on trastuzumab.

B cell translocation gene 1 contributes to antisense Bcl-2-mediated apoptosis in breast cancer cells.

The antiapoptotic protein Bcl-2 is overexpressed in a majority of breast cancers, and is associated with a diminished apoptotic response and resistance to various antitumor agents. Bcl-2 inhibition is currently being explored as a possible strategy for sensitizing breast cancer cells to standard chemotherapeutic agents. Antisense Bcl-2 oligonucleotides represent one method for blocking the antiapoptotic effects of Bcl-2. In this study, we show that antisense Bcl-2 efficiently blocks Bcl-2 expression, resulting in the apoptosis of breast cancer cells. Antisense Bcl-2-mediated cytotoxicity was associated with the induction of the B cell translocation gene 1 (BTG1). Importantly, knockdown of BTG1 reduced antisense Bcl-2-mediated cytotoxicity in breast cancer cells. Furthermore, BTG1 expression seems to be negatively regulated by Bcl-2, and exogenous expression of BTG1 induced apoptosis. These results suggest that BTG1 is a Bcl-2-regulated mediator of apoptosis in breast cancer cells, and that its induction contributes to antisense Bcl-2-mediated cytotoxic effects.