Interferon regulatory factor-1 regulates reconstituted extracellular matrix (rECM)-mediated apoptosis in human mammary epithelial cells.

Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the gamma-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex.

Suppression of p53 function in normal human mammary epithelial cells increases sensitivity to extracellular matrix-induced apoptosis.

Little is known about the fate of normal human mammary epithelial cells (HMECs) that lose p53 function in the context of extracellular matrix (ECM)-derived growth and polarity signals. Retrovirally mediated expression of human papillomavirus type 16 (HPV-16) E6 and antisense oligodeoxynucleotides (ODNs) were used to suppress p53 function in HMECs as a model of early breast cancer. p53+ HMEC vector controls grew exponentially in reconstituted ECM (rECM) until day 6 and then underwent growth arrest on day 7. Ultrastructural examination of day 7 vector controls revealed acinus-like structures characteristic of normal mammary epithelium. In contrast, early passage p53- HMEC cells proliferated in rECM until day 6 but then underwent apoptosis on day 7. p53- HMEC-E6 passaged in non-rECM culture rapidly (8-10 passages), lost sensitivity to both rECM-induced growth arrest and polarity, and also developed resistance to rECM-induced apoptosis. Resistance was associated with altered expression of alpha3-integrin. Treatment of early passage p53- HMEC-E6 cells with either alpha3- or beta1-integrin function-blocking antibodies inhibited rECM-mediated growth arrest and induction of apoptosis. Our results indicate that suppression of p53 expression in HMECs by HPV-16 E6 and ODNs may sensitize cells to rECM-induced apoptosis and suggest a role for the alpha3/beta1-heterodimer in mediating apoptosis in HMECs grown in contact with rECM.

Suppression of pRB expression in normal human mammary epithelial cells is associated with resistance to all-trans-retinoic acid but not N-(4-hydroxylphenyl)-retinamide.

Despite the widespread clinical use of synthetic and naturally occurring retinoids, the down stream targets of retinoids have not been fully characterized. We observe that G(1/0)-phase arrest induced by all-trans-retinoic acid (ATRA) in normal human mammary epithelial cells (HMECs) is temporally associated with a significant decrease in the levels of hyperphosphorylated retinoblastoma protein (pRB). Suppression of pRB protein expression in HMECs by retroviral-mediated expression of the E7 protein of the human papillomavirus strain 16 (HPV-16) was associated with resistance to ATRA-mediated growth arrest but not to the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR or fenretinide). 4-HPR but not ATRA induced apoptosis in HMECs independent of the level of pRB protein expression. These observations suggest that ATRA- but not 4-HPR-mediated growth arrest may be dependent on the coordinated expression of pRB and emphasize the chemotherapeutic potential of 4-HPR, particularly for suppressing growth of tumors lacking pRB function.

Human papillomavirus type 16 E6 inactivation of p53 in normal human mammary epithelial cells promotes tamoxifen-mediated apoptosis.

Aberrant p53 expression is frequently observed in mammary epithelial cells obtained from women at high risk for developing breast cancer and is a predictor for the subsequent development of malignancy. Tamoxifen has recently been shown to reduce the incidence of noninvasive breast cancer in high-risk women, but the molecular mechanism of tamoxifen chemoprevention in mammary epithelial tissue that does not overexpress the estrogen receptor is poorly understood. We suppressed p53 expression by retroviral-mediated expression of human papillomavirus type-16 E6 protein (HPV-16 E6) in human mammary epithelial cells (HMECs) to develop an in vitro model of tamoxifen chemoprevention in the context of p53 loss. Early passage p53(-) HMEC-E6-transduced cells treated with 1.0 microM tamoxifen rapidly underwent apoptosis. In contrast, early passage p53(+) HMEC-LXSN vector controls treated with 1.0 microM tamoxifen underwent G1-G0-phase arrest but did not undergo apoptosis. p53(-) HMEC-E6 cells rapidly acquired resistance to tamoxifen-mediated apoptosis after 10 passages in culture (in the absence of tamoxifen). Both p53(+) and p53(-) HMECs exhibited a low level of estrogen receptor staining and minimal estrogen binding, characteristic of proliferating normal luminal mammary epithelial cells. Tamoxifen-mediated apoptosis in p53(-) HMEC-E6 cells was not blocked by inhibitors of transcription and protein synthesis. These data suggest that the acute loss of p53 function in HMECs by expression of HPV-16 E6 results in marked sensitivity to tamoxifen-mediated apoptosis but that resistance to apoptosis rapidly develops within 10 passages in vitro. Observations in our model system predict a critical role for the early institution of tamoxifen chemoprevention.

Tamoxifen but not 4-hydroxytamoxifen initiates apoptosis in p53(-) normal human mammary epithelial cells by inducing mitochondrial depolarization.

Despite the widespread clinical use of tamoxifen as a breast cancer prevention agent, the molecular mechanism of tamoxifen chemoprevention is poorly understood. Abnormal expression of p53 is felt to be an early event in mammary carcinogenesis. We developed an in vitro model of early breast cancer prevention to investigate how tamoxifen and 4-hydroxytamoxifen may act in normal human mammary epithelial cells (HMECs) that have acutely lost p53 function. p53 function was suppressed by retrovirally mediated expression of the human papillomavirus type 16 E6 protein. Tamoxifen, but not 4-hydroxytamoxifen, rapidly induced apoptosis in p53(-) HMEC-E6 cells as evidenced by characteristic morphologic changes, annexin V binding, and DNA fragmentation. We observed that a decrease in mitochondrial membrane potential, mitochondrial condensation, and caspase activation preceded the morphologic appearance of apoptosis in tamoxifen-treated early passage p53(-) HMEC-E6 cells. p53(-) HMEC-E6 cells rapidly developed resistance to tamoxifen-mediated apoptosis within 10 passages in vitro. Resistance to tamoxifen in late passage p53(-) HMEC-E6 cells correlated with an increase in mitochondrial mass and a lack of mitochondrial depolarization and caspase activation following tamoxifen treatment. We hypothesize that an early event in the induction of apoptosis by tamoxifen involves mitochondrial depolarization and caspase activation, and this may be important for effective chemoprevention.

Dysregulated expression of cyclin D1 in normal human mammary epithelial cells inhibits all-trans-retinoic acid-mediated G0/G1-phase arrest and differentiation in vitro.

Overexpression of cyclin D1 protein is observed in the majority of breast cancers, suggesting that dysregulated expression of cyclin D1 might be a critical event in breast cancer carcinogenesis. We investigated whether retroviral-mediated expression of cyclin D1 might affect all-trans-retinoic acid (ATRA)-mediated growth inhibition and differentiation of normal cultured human mammary epithelial cells (HMECs). HMECs treated with 1.0 microM ATRA undergo irreversible growth inhibition starting at 24 h and complete G0/G1-phase arrest by Day 3. Cyclin D1 protein levels are observed to decrease in association with the initiation of growth arrest starting at 24 h and then increase by approximately 35% on Day 3. Concomitant with this observed increase in cyclin D1, HMECs undergo morphologic changes consistent with progression to a more differentiated phenotype, including an increase in cell size, increased cell spreading, increased tonofilaments, and accumulation of cytoplasmic vesicles containing lipid. Dysregulated expression of cyclin D1 in HMECs results in inhibition of G0/G1-phase arrest mediated by ATRA. In addition, HMECs expressing exogenous cyclin D1 are resistant to differentiation by ATRA. Our results suggest that coordinated expression of cyclin D1 may be critical for normal mammary epithelial cell homeostasis, and dysregulated expression of cyclin D1 might result in retinoid resistance and promote mammary carcinogenesis.

Retinoic acid-mediated G1-S-phase arrest of normal human mammary epithelial cells is independent of the level of p53 protein expression.

Retinoids mediate the normal growth of a variety of epithelial cells and may play an important role in the chemoprevention of breast cancer. Despite the widespread clinical use of retinoids, specific target genes that are regulated by retinoids are relatively poorly characterized. We reported previously that all-trans-retinoic acid (ATRA) mediates G1-S-phase arrest in normal human mammary epithelial cells (HMECs). The tumor suppressor gene p53 is thought to be a critical regulator of G1-S-phase arrest mediated by DNA-damaging agents such as chemotherapy and radiation. The role of p53 protein expression in G1-S-phase arrest mediated by the differentiating agent ATRA is unknown. Increased expression of p53 protein is observed in ATRA-treated HMECs at 72 h; however, initiation of G1-S-phase arrest starts at 24 h, suggesting that this observed induction of p53 is a secondary event. Using retroviral-mediated gene transfer, we expressed the E6 protein of the human papillomavirus strain 16 (HPV-16) in HMECs. The HPV-16 E6 protein binds to p53 and targets it for degradation. Western analysis confirmed that HPV-16 E6-transduced HMECs had markedly decreased levels of p53 protein expression. Suppression of cellular p53 levels in HMECs did not alter the sensitivity of HMECs to ATRA-mediated growth arrest. Our studies suggest that ATRA-mediated G1-S-phase arrest is independent of the level of p53 protein expression. We also tested the ability of estrogen and antiestrogens to induce growth arrest in HMECs lacking p53 expression and found no decrease in the sensitivity of these cells to these agents. Our results emphasize the chemotherapeutic potential of ATRA and antiestrogens, particularly for suppressing the growth of tumors lacking functional p53.

A retrospective review of paclitaxel-associated gastrointestinal necrosis in patients with epithelial ovarian cancer.

Seven patients with gastrointestinal necrosis following paclitaxel chemotherapy are reported. Four of seven patients had platinum refractory disease, while 3/7 patients received primary paclitaxel therapy. Complications occurred 5 to 16 days following paclitaxel therapy. The most common clinical presentation was fever (7/7 patients), neutropenia (6/7 patients), and abdominal pain (6/7 patients). All seven patients developed gastrointestinal necrosis following the first cycle of paclitaxel chemotherapy. The exact mechanism by which this complication occurs is poorly understood. We postulate that gastrointestinal necrosis may be the result of a direct drug effect on the gastrointestinal epithelium and might involve a synergistic interaction between compromised bowel and paclitaxel-induced mitotic arrest. We observe that the incidence of gastrointestinal necrosis in patients with platinum refractory disease is 4 of 108 patients (3.7%). The incidence of this complication in patients receiving primary paclitaxel at our institution is 3 of approximately 128 patients (2.3%). Eighteen cases to date have been identified in the literature. A high index of suspicion of this complication should be considered for patients presenting with neutropenic fever and abdominal pain following paclitaxel chemotherapy.

Inhibition of retinoic acid receptor function in normal human mammary epithelial cells results in increased cellular proliferation and inhibits the formation of a polarized epithelium in vitro.

The expression of retinoic acid receptor-beta (RAR beta) mRNA is absent or down-regulated in a majority of breast cancers, suggesting that loss of retinoic acid receptor function may be a critical event in breast cancer carcinogenesis. We developed an in vitro system to investigate whether the loss of retinoic acid receptor (RAR) function might affect the proliferation and structural differentiation of normal cultured human mammary epithelial cells (HMECs). Utilizing a truncated retinoic acid receptor (RAR)-alpha construct exhibiting dominant-negative activity against retinoic acid receptor isoforms alpha, beta, and gamma (DNRAR), we inhibited normal retinoic acid receptor function in HMECs. Suppression of RAR function in HMECs resulted in reduced growth inhibition mediated by all-trans-retinoic acid (ATRA). Moreover, the doubling time of HMECs expressing the DNRAR was significantly shortened, associated with a decrease in the percentage of cells in G1 and an increase in the percentage of cells in S-phase relative to controls. In addition, HMECs expressing the DNRAR cultured in prepared extracellular matrix exhibited a loss of extracellular matrix-induced growth arrest and formation of a polarized ductal epthelium. Our results suggest that ATRA and RARs may play an important role in regulating the proliferation of HMECs and in promoting differentiation.

All-trans-retinoic acid mediates G1 arrest but not apoptosis of normal human mammary epithelial cells.

Retinoids mediate the normal growth of a variety of epithelial cells and may play an important role in the chemoprevention of certain malignancies. Loss of retinoic acid (RA) receptor-beta function may be an important event in mammary carcinogenesis, because the majority of breast cancers, in contrast to normal mammary epithelial cells, fail to express this receptor. We previously reported that all-trans-RA mediates G1 arrest as well as apoptosis in certain RAR beta-transduced breast cancer cell lines. We now report the effect of RA on normal human mammary epithelial cells (HMECs), which express functionally active retinoid receptors. We observe that RA induces growth suppression and G1 arrest of these HMECs but find no evidence that RA mediates apoptosis in these normal cell strains. This RA-induced G1 arrest is temporally associated with decreased levels of hyperphosphorylated retinoblastoma protein without any significant changes in c-myc, p53, p21, or p27 expression. Expression of cyclin D1, cyclin-dependent kinase 4, and cyclin E proteins, however, decreased in association with RA-mediated G1 arrest. Our studies suggest that growth inhibition, rather than apoptosis, may be a mechanism by which RA and RA receptors act to prevent the malignant transformation of normal mammary epithelial cells. The molecular target(s) of the activated RA receptors that mediate this G1 arrest in HMECs appear to be associated with a retinoblastoma-dependent pathway.

Expression of retinoic acid receptor beta mediates retinoic acid-induced growth arrest and apoptosis in breast cancer cells.

The expression of the retinoic acid receptor beta (RAR beta) mRNA is absent or down-regulated in most human breast cancer cell lines. To investigate the role RAR beta may have in regulating the proliferation of breast cancer cells, we used retroviral vector-mediated gene transduction to introduce the human RAR beta gene into two RAR beta-negative breast tumor cell lines, MCF-7 and MDA-MB-231. RAR beta-transduced clones underwent growth inhibition associated with G1 arrest when treated with 1 microM all-trans-retinoic acid (RA). Moreover, the MCF7-RAR beta transduced clones also underwent apoptosis after 4 to 6 days of RA treatment. The RA-induced growth arrest in MDA231-RAR beta transduced cells is associated with c-myc mRNA down-regulation, whereas the RA-mediated apoptosis of MCF7-RAR beta transduced cells is not associated with c-myc down-regulation. These observations suggest a critical role for RAR beta in mediating growth arrest and apoptosis in breast cancer cells.

Primary central nervous system recurrence after paclitaxel therapy for epithelial ovarian malignancy.

Paclitaxel is currently being utilized to treat neoplasms which have a significant incidence of central nervous system metastases. It is, however, unclear as to whether paclitaxel crosses the blood-brain barrier. In this report, the authors describe a patient with refractory epithelial ovarian cancer treated with paclitaxel at 135 mg/m2/24 hr every 21 days. The patient achieved a complete clinical response of all abdominal and pelvic disease, but simultaneously developed central nervous system metastases. Paclitaxel was effective against the patient's abdominal and pelvic disease but was not protective against central nervous system metastasis.

Paclitaxel (Taxol) treatment for refractory ovarian cancer: phase II clinical trial.

OBJECTIVE: Our aim was to determine the efficacy and toxicity of paclitaxel in the treatment of refractory and platinum-resistant epithelial ovarian cancer. STUDY DESIGN: Eligibility required three prior failed chemotherapy regimens and documented platinum resistance. One hundred patients with advanced ovarian cancer received paclitaxel 135 mg/m2 over 24 hours every 21 days with optional granulocyte colony-stimulating factor support. RESULTS: Paclitaxel was generally well tolerated. In four patients bowel perforation or fistula developed. After three cycles 34% of patients had stable disease and 25% of patients demonstrated a response, either partial or complete. After six cycles 24% of patients continued to respond. To date, six patients have achieved a complete response. CONCLUSION: A 25% response rate in patients with refractory ovarian cancer was observed, which was durable to six cycles.