Beatrice Uziely

Liposomal doxorubicin: antitumor activity and unique toxicities during two complementary phase I studies.

PURPOSE The purpose of our studies was to define the maximal-tolerated dose of liposomal doxorubicin (DOX-SL; Liposome Technology Inc, Menlo Park, CA), a doxorubicin formulation of polyethyleneglycol-coated liposomes, characterize the toxicities associated with this formulation, and evaluate any indication of antitumor activity within a phase I setting. PATIENTS AND METHODS Two separate phase I studies were conducted following the initial human pharmacokinetic testing at one of the sites (Hadassah). The starting dose of 20 mg/m2 at the University of Southern California was just below the dose without toxicity in the pharmacokinetic study. At Hadassah, the phase I starting dose was just above their earlier safe single doses, 60 mg/m2. Both studies involved cohorts of at least three patients and redosing every 3 to 4 weeks. To determine the recommended dose for phase II trials, an additional level of 50 mg/m2 every 3 weeks was explored, and the level of 60 mg/m2 every 4 weeks was expanded. RESULTS A total of 56 patients receiving 281 courses of DOX-SL was accrued and evaluated for toxicity. Hand-foot (H-F) syndrome and stomatitis are the two main dose-limiting factors of DOX-SL. Stomatitis was dose-limiting for high single doses of DOX-SL greater than 70 mg/m2. Skin toxicity manifested primarily as H-F syndrome was dose-limiting for repetitive dosing, but acceptable at either 50 mg/m2 every 3 weeks or 60 mg/m2 every 4 weeks. Attenuation of acute subjective symptoms and lack of alopecia were generally observed. Patients with carcinomas of the breast, ovary, prostate, and head and neck were among those showing objective antitumor responses or improvement based, in part, on blood levels of tumor markers. CONCLUSION The toxicity profile of DOX-SL differs prominently from that of the free drug administered by bolus or rapid infusion and with some differences, resembles that of prolonged continuous infusion. This finding, as well as the antitumor activity observed, supports wide phase II testing of DOX-SL in solid tumors.

CLINICAL STUDIES OF LIPOSOME-ENCAPSULATED DOXORUBICIN

Initial clinical studies with doxorubicin entrapped in the bilayer of phosphatidylglycerol-rich liposomes were hindered by the avid reticuloendothelial system (RES) uptake and by drug leakage from circulating liposomes. In contrast, recent tests of a doxorubicin formulation of polyethyleneglycol-coated liposomes (Doxil) in cancer patients indicate that the drug pharmacokinetic properties are significantly altered, with a prolonged distribution half-life of approximately 2 days. Plasma fractionation studies show that nearly all the drug measured in plasma is in liposome-encapsulated form. The dose of Doxil has been escalated from 25 to 60 mg/m2. Stomatitis is the most significant toxicity, and skin toxicity, in the form of hand-foot syndrome, may complicate the repeated administration of Doxil. A number of objective antitumor responses in a variety of malignancies have been observed, indicating that Doxil is an active antitumor compound. Polyethyleneglycol-coated liposomes show a distinct advantage over previous liposome formulations directed at the RES and appear to be a promising drug delivery system for doxorubicin.

The effect of tamoxifen on the endometrium

SummaryTamoxifen is one of the most important treatments for breast cancer, especially in postmenopausal patients. It acts primarily as an anti-estrogenic agent, due to its cytoplasmic estrogen receptor binding capacity. However, it also exerts a mild estrogenic effect. Since the prolonged use of estrogen has been reported to increase the rate of benign and malignant changes in the endometrium, we evaluated whether there is a correlation between tamoxifen therapy and endometrial benign and malignant conditions. The study group comprised 95 patients with breast cancer who were treated with tamoxifen. No control group was examined. Patients underwent vaginal ultrasonography and endometrial biopsy in order to evaluate any changes in the endometrium occurring during tamoxifen therapy. Pathological changes were observed in 14 patients, 13 of whom were treated with tamoxifen for more than 12 months. Of these women, 3 were diagnosed with endometrial cancer, 3 had mild dysplasia, 3 had endometrial hyperplasia, and 4 had a benign endometrial polyp. Our findings indicate a significant correlation between long-term tamoxifen administration and endometrial proliferation. We therefore recommend that women treated with tamoxifen for more than 12 months have an annual vaginal ultrasonography and endometrial biopsy.

Human Protease-Activated Receptor 1 Expression in Malignant Epithelia: A Role in Invasiveness

While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.

Ovarian cysts in premenopausal and postmenopausal tamoxifen-treated women with breast cancer

OBJECTIVE Our purpose was to investigate the frequency of ovarian cysts in tamoxifen-treated breast cancer patients. STUDY DESIGN The study population included 95 consecutive tamoxifen-treated premenopausal and postmenopausal women with breast cancer who were followed up by the outpatient clinic at the Hadassah University Hospital between September 1990 and June 1992. Tamoxifen was administered orally (20 mg/day). All patients underwent a pelvic examination and vaginal ultrasonography with a 5 MHz vaginal probe. RESULTS During the study 11 of 95 tamoxifen-treated breast cancer patients (11%) had ovarian cysts. Five cysts were detected in postmenopausal women (6.3% of the postmenopausal women) and six in premenopausal women (37.5% of the premenopausal women). In postmenopausal and premenopausal women the mean tamoxifen treatment interval was 19.4 +/- 7.8 months (range 4 to 48 months) and 28 +/- 6.1 months (range 12 to 54 months), respectively (p = 0.41). In 8 of the 11 patients the ovarian cystic enlargement disappeared after cessation of tamoxifen treatment. Two patients underwent laparotomy because of persistent cysts and the third because of a rapidly growing myoma. The three cysts were found to be benign. CONCLUSION Ovarian cysts are a common side effect of tamoxifen treatment. The ovarian cysts can develop in tamoxifen-treated premenopausal as well as postmenopausal women with breast cancer. Most of the tamoxifen-associated cysts disappear after tamoxifen treatment is abandoned.

G Protein-Coupled Receptors in Cancer

Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

Protease-Activated Receptor-1 (hPar1), A Survival Factor Eliciting Tumor Progression

Although ample evidence point to the central involvement of protease activated receptor-1 (PAR1) in tumor progression, little is known about the fate of the tumor when hPar1 is being silenced. We observed that hPar1 antisense clones exhibit low PAR1 levels, attenuated cell proliferation and invasion in vitro, and tumor formation in vivo. These clones showed noticeably reduced paxillin phosphorylation compared with the parental A375SM cells, whereas no change in the integrin levels was noticed. Antisense clones injected into the mice resulted in very few and only occasional small tumors, whereas advanced and vascularized tumors were observed in A375SM cells. The antisense-derived tumor sections expressed active caspase-3, increased terminal deoxynucleotidyl transferase–mediated nick-end labeling staining, and a markedly reduced proliferating cell nuclear antigen level compared with A375SM cell–derived tissue sections. Likewise, ablation of the hPar1 gene in a tetracycline-inducible hPar1 system leads to apoptosis in immature blood vessels, whereas mature vessels were unaffected. The activation of PAR1-induced pAkt/protein kinase B abrogated serum-deprived BimEL induction and also markedly inhibited Bax levels. On the other hand, small interfering RNA silencing of the hPar1 gene induced the expression of BimEL, a direct substrate of Akt/protein kinase B and also induced expression of active caspase-9 and caspase-3. These results altogether identify PAR1 as a survival factor that protects cells from undergoing apoptosis. We conclude that whereas PAR1 gene expression correlates with tumor progression, its neutralization effectively initiates an apoptotic pathway leading at least in part to significantly reduced tumor formation. (Mol Cancer Res 2007;5(3):229–40)

PAR1 plays a role in epithelial malignancies: transcriptional regulation and novel signaling pathway.

Protease‐activated receptor1 (PAR1) is the first and prototype member of an established PAR family comprising four members. The role of PAR1 in tumor biology has been established, and is characterized by a consistent direct correlation between overexpression of its levels and epithelial tumor aggressiveness. We have found that high expression of the human Par1 (hPar1) gene in epithelial tumors is controlled largely at the transcriptional level. This led us to assign Egr‐1, a transcription activator, as an inducer of hPar1, and p53, a tumor suppressor gene, as an inhibitor, both acting to achieve fine tuning of hPar1 in prostate carcinoma. High PAR1 levels maintain prosurvival signals in tumor cells while silencing or ablation of the gene induce apoptosis. Studies of our hPar1 transgenic mice, which overexpress hPar1 in the mammary glands, revealed a novel PAR1‐induced β‐catenin stabilization function. The components connecting PAR1 to β‐catenin stabilization have been determined, assigning at first Gα13 as a selective immediate component. The PAR1‐Gα13 axis recruits disheveled (DVL), an upstream signaling partner of the canonical Wnt signaling pathway. Silencing of DVL by siRNA‐DVL potently abrogates PAR1‐induced β‐catenin stabilization, demonstrating its critical role in the process. We, thus, propose that transcriptional regulation of hPar1 gene over expression in epithelia malignancies initiates a novel signaling pathway, directly connecting to β‐catenin stabilization, a core event in both tumorigenesis and developmental processes.

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

Background While protease-activated-receptor 1 (PAR1) plays a central role in tumor progression, little is known about the cell signaling involved. Methodology/Principal Findings We show here the impact of PAR1 cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR1 activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR1 C-tail did not prevent Shc-PAR1 association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR1 C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR1-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR1 -C-tail, hPar1 oncogenic properties are abrogated. Conclusions/Significance This is the first demonstration that a cytoplasmic portion of the PAR1 C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR1 -associating region in the breast cancer signaling niche.

The relationship between symptom severity and symptom interference, education, age, marital status, and type of chemotherapy treatment in Israeli women with early-stage breast cancer.

PURPOSE/OBJECTIVES To examine symptom severitys relationship to symptom interference, education, age, marital status, and type of chemotherapy treatment in Israeli women with stage I or II breast cancer. DESIGN Cross-sectional, descriptive, correlational design. SETTING Hadassah University Hospitals oncology daytime care unit in Israel. SAMPLE 51 women with stage I or II breast cancer who were receiving an adjuvant chemotherapy protocol that included doxorubicin. METHODS Women receiving adjuvant chemotherapy were given the M.D. Anderson Symptom Inventory (MDASI), a modified version of the Breast Cancer Prevention Trial Hot Flashes Subscale (BCPT-HFS), and a demographic and treatment questionnaire to assess their symptoms toward the end of their chemotherapy treatment. MAIN RESEARCH VARIABLES Symptom severity, symptom interference, education, age, marital status, and type of chemotherapy treatment. FINDINGS The most frequent and severe symptoms were fatigue, sleep disturbance, and drowsiness. The MDASI symptom severity total scores were positively correlated with total scores of interference with activities of daily life, with most individual symptoms being significantly related to the total interference scores. The strongest relationships were found with fatigue, distress, and sadness. Education was inversely related to the MDASI general symptom severity total scores; age was inversely related to the BCPT-HFS total scores. Patients who received treatment with doxorubicin plus cyclophosphamide or doxorubicin, cyclophosphamide, plus fluorouracil had greater symptom severity than those who received doxorubicin plus cyclophosphamide followed by paclitaxel and had their symptoms evaluated after receiving paclitaxel. CONCLUSIONS Increased symptom severity disrupts daily function and life in women with breast cancer. IMPLICATIONS FOR NURSING Evidence-based symptom profiles for different chemotherapy protocols are needed.