TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells.

Metastatic growth in breast cancer (BC) has been proposed as an exclusive property of cancer stem cells (CSCs). However, formal proof of their identity as cells of origin of recurrences at distant sites and the molecular events that may contribute to tumor cell dissemination and metastasis development are yet to be elucidated. In this study, we analyzed a set of patient-derived breast cancer stem cell (BCSC) lines. We found that in vitro BCSCs exhibit a higher chemoresistance and migratory potential when compared with differentiated, nontumorigenic, breast cancer cells (dBCCs). By developing an in vivo metastatic model simulating the disease of patients with early BC, we observed that BCSCs is the only cell population endowed with metastatic potential. Gene-expression profile studies comparing metastagenic and non-metastagenic cells identified TAZ, a transducer of the Hippo pathway and biomechanical cues, as a central mediator of BCSCs metastatic ability involved in their chemoresistance and tumorigenic potential. Overexpression of TAZ in low-expressing dBCCs induced cell transformation and conferred tumorigenicity and migratory activity. Conversely, loss of TAZ in BCSCs severely impaired metastatic colonization and chemoresistance. In clinical data from 99 BC patients, high expression levels of TAZ were associated with shorter disease-free survival in multivariate analysis, thus indicating that TAZ may represent a novel independent negative prognostic factor. Overall, this study designates TAZ as a novel biomarker and a possible therapeutic target for BC.

Selective GPER activation decreases proliferation and activates apoptosis in tumor Leydig cells.

We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients.

Checkpoint kinase 1 inhibitors for potentiating systemic anticancer therapy.

The checkpoint kinase 1 (Chk1) is a key component of the DNA damage response, a molecular network deputed to maintain genome integrity. Nevertheless, cancer cells aberrantly exploit these circuits to overcome chemotherapy-induced cytotoxicity. Chk1 inhibitors have been developed as a chemopotentiating strategy and different molecular mechanisms underlying the synergism with chemotherapeutics have been uncovered. The monotherapy with Chk1 inhibitors seems to be endowed with antitumor activity against cancer cells characterized by specific defects in the DNA damage machinery or characterized by elevated levels of oncogene-induced replication stress. In this biological framework Chk1 neutralization represents a synthetic lethality-based therapeutic approach. Moreover, a dual targeting of the DNA damage machinery has been proposed envisioning the association of Chk1 abrogation with poly-ADP ribose polymerase inhibitors. The spectrum of antitumor properties of Chk1 antagonists is completed by the activity against cancer stem cells, the prominent tumorigenic population that is equipped to survive stressful conditions through multiple and interconnected mechanisms. Although the clinical development of the first generation of Chk1 antagonists was hindered by off-target effects and an unfavorable pharmacokinetic profile, a new wave of early clinical trials with more selective compounds are currently being carried out. To this end, the identification of predictive biomarkers and an in-depth characterization of molecular circuits governed by Chk1 are issues that need to be addressed for sharpening the therapeutic potential of Chk1 inhibitors.

Therapeutic targeting of Chk1 in NSCLC stem cells during chemotherapy.

Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

Role of estrogen receptor alpha in modulating IGF-I receptor signaling and function in breast cancer.

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is a multifunctional transmembrane tyrosine kinase that has been implicated in neoplastic transformation. The tumorigenic potential of IGF-IR relies on its strong anti-apoptotic and mitogenic activity. The growth and survival signals of IGF-IR are mediated through multiple intracellular pathways, many of which emanate from insulin receptor substrate 1 (IRS-1). In hormone-dependent breast cancer cells, IGF-IR and IRS-1 are often co-expressed with the estrogen receptor alpha (ERalpha), and IGF-I and ER systems are engaged in a powerful functional cross-talk. Most notably, activation of ERalpha upregulates the expression of IRS-1, IGF-IR, and IGF-1, which results in amplification of IGF-I responses. Reciprocally, stimulation of IGF-IR increases the phosphorylation and activity of ERalpha. In contrast, in ERalpha-negative breast cancer cells and tumors, the levels of IGF-IR and IRS-1 are often decreased and IGF-I is non-mitogenic. Our data suggest that defective IGF-IR signaling in ERalpha-negative cells is related, at least in part, to improper activation of the IRS-1/PI-3K/Akt/GSK-3 pathway and lack of Rb1 phosphorylation. These defects are partially reversed by re-expression of ERalpha. Interestingly, some non-mitogenic IGF-I responses, such as migration and invasion are retained in the absence of ERalpha, suggesting that IGF-IR function in breast cancer cells might depend on the ERalpha status. The understanding of how ERalpha may dictate IGF-I responses will help in devising rational anti-IGF-IR strategies for breast cancer treatment.

Differential insulin-like growth factor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells.

The insulin-like growth factor I receptor (IGF-IR) is a ubiquitous and multifunctional tyrosine kinase that has been implicated in breast cancer development. In estrogen receptor (ER)-positive breast tumors, the levels of the IGF-IR and its substrate, insulin-receptor substrate 1 (IRS-1), are often elevated, and these characteristics have been linked with increased radioresistance and cancer recurrence. In vitro, activation of the IGF-IR/IRS-1 pathway in ER-positive cells improves growth and counteracts apoptosis induced by anticancer treatments. The function of the IGF-IR in hormone-independent breast cancer is not clear. ER-negative breast cancer cells often express low levels of the IGF-IR and fail to respond to IGF-I with mitogenesis. On the other hand, anti-IGF-IR strategies effectively reduced metastatic potential of different ER-negative cell lines, suggesting a role of this receptor in late stages of the disease. Here we examined IGF-IR signaling and function in ER-negative MDA-MB-231 breast cancer cells and their IGF-IR-overexpressing derivatives. We demonstrated that IGF-I acts as a chemoattractant for these cells. The extent of IGF-I-induced migration reflected IGF-IR levels and required the activation of phosphatidylinositol 3-kinase (PI-3K) and p38 kinases. The same pathways promoted IGF-I-dependent motility in ER-positive MCF-7 cells. In contrast with the positive effects on cell migration, IGF-I was unable to stimulate growth or improve survival in MDA-MB-231 cells, whereas it induced mitogenic and antiapoptotic effects in MCF-7 cells. Moreover, IGF-I partially restored growth in ER-positive cells treated with PI-3K and ERK1/ERK2 inhibitors, whereas it had no protective effects in ER-negative cells. The impaired IGF-I growth response of ER-negative cells was not caused by a low IGF-IR expression, defective IGF-IR tyrosine phosphorylation, or improper tyrosine phosphorylation of IRS-1. Also, the acute (15-min) IGF-I activation of PI-3 and Akt kinases was similar in ER-negative and ER-positive cells. However, a chronic (2-day) IGF-I exposure induced the PI-3K/Akt pathway only in MCF-7 cells. The reactivation of this pathway in ER-negative cells by overexpression of constitutively active Akt mutants was not sufficient to significantly improve proliferation or survival (with or without IGF-I), which indicated that other pathways are also required to support these functions. Our results suggest that in breast cancer cells, IGF-IR can control nonmitogenic processes regardless of the ER status, whereas IGF-IR growth-related functions may depend on ER expression.

IGF-I receptor-induced cell-cell adhesion of MCF-7 breast cancer cells requires the expression of junction protein ZO-1.

Hyperactivation of the insulin-like growth factor I receptor (IGF-IR) contributes to primary breast cancer development, but the role of the IGF-IR in tumor metastasis is unclear. Here we studied the effects of the IGF-IR on intercellular connections mediated by the major epithelial adhesion protein, E-cadherin (E-cad). We found that IGF-IR overexpression markedly stimulated aggregation in E-cad-positive MCF-7 breast cancer cells, but not in E-cad-negative MDA-MB-231 cells. However, when the IGF-IR and E-cad were co-expressed in MDA-MB-231 cells, cell-cell adhesion was substantially increased. The IGF-IR-dependent cell-cell adhesion of MCF-7 cells was not related to altered expression of E-cad or alpha-, beta-, or gamma-catenins but coincided with the up-regulation of another element of the E-cad complex, zonula occludens-1 (ZO-1). ZO-1 expression (mRNA and protein) was induced by IGF-I and was blocked in MCF-7 cells with a tyrosine kinase-defective IGF-IR mutant. By co-immunoprecipitation, we found that ZO-1 associates with the E-cad complex and the IGF-IR. High levels of ZO-1 coincided with an increased IGF-IR/alpha-catenin/ZO-1-binding and improved ZO-1/actin association, whereas down-regulation of ZO-1 by the expression of an anti-ZO-1 RNA inhibited IGF-IR-dependent cell-cell adhesion. The results suggested that one of the mechanisms by which the activated IGF-IR regulates E-cad-mediated cell-cell adhesion is overexpression of ZO-1 and the resulting stronger connections between the E-cad complex and the actin cytoskeleton. We hypothesize that in E-cad-positive cells, the IGF-IR may produce antimetastatic effects.

Kidney transplantation: state of the art.

Despite ever-improving health care and new advances in medical technology, the number of Americans who develop end-stage renal disease continues to increase. Diabetes remains the leading cause of new cases, followed by hypertension and glomerulonephritis. More than 200,000 patients require dialysis and more than 40,000 are awaiting kidneys for transplantation. Kidney transplantation has been extremely successful with 1-year patient and graft survival rates at 95% and 90%, respectively. The advantages of kidney transplantation are reversal of many of the pathophysiologic changes associated with renal failure as normal kidney function is restored, elimination of dependence on dialysis and the associated dietary restrictions, the opportunity to return to normal life activities, and lower medical costs than dialysis after the first year. The shortage of donor kidneys is the major limiting factor. Because of the supply and demand discrepancy, maximum use of donors from all sources, appropriate recipient selection, and equitable allocation are critical.

SHC-alpha5beta1 integrin interactions regulate breast cancer cell adhesion and motility.

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with alpha5beta1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.

Issues surrounding xenotransplantation.

Currently there is a shortage of cadaver organs that can be transplanted from one human being to another. In response to this shortage, scientists and medical researchers have developed techniques for transplanting animal organs into humans, a procedure known as xenotransplantation. This may address the shortage of organs for patients in need; however, it raises other concerns related to cross-species disease transmission, consent issues, ethical issues of sacrificing animals for their organs, psychological issues of receiving organs from an animal, religious concerns, and economic factors. These medical, ethical, and philosophical issues need to be thoroughly addressed before xenotransplantation becomes a reality.

Azathioprine monotherapy in HLA-identical live donor kidney transplant recipients.

The high success rate of HLA-identical sibling transplants and our previous experience with steroid-free immunosuppressive regimens and cyclosporine withdrawal prompted us to evaluate the safety and efficacy of monotherapy with azathioprine in 12 HLA-identical kidney transplant recipients with a serum creatinine concentration less than 176.8 mumol/L, a 1-way stimulatory index less than 2.0 in a post-transplant mixed lymphocyte culture, and a demonstrated tolerance of a minimum azathioprine dose of 1.0 mg/kg per day without leukopenia. Eleven of 12 patients were successfully converted to azathioprine monotherapy without a significant change in serum creatinine concentration for as long as 76 months. Benefits of steroid and cyclosporine withdrawal included a significant reduction in mean systolic and diastolic blood pressure, number of blood pressure medications, total serum cholesterol, and glycohemoglobin in diabetic subjects. Our results suggest that azathioprine monotherapy is safe and effective in a select group of HLA-identical sibling transplants, but these benefits must be carefully balanced against an associated risk of precipitating acute allograft rejection.

Issues in cyclosporine drug substitution: implications for patient management.

Substantial improvements in short-term and long-term outcomes for kidney transplant recipients have resulted from better use of existing immunosuppressive agents and newer treatment options. Calcineurin inhibitors (e.g., cyclosporine and tacrolimus) remain the foundation of immunosuppressive therapy. These agents are considered critical-dose drugs because of their narrow therapeutic range, variable pharmacokinetics, formulation-dependent bioavailability, and negative clinical consequences of underdosing or overdosing. With the recent introduction of a new cyclosporine formulation, concern exists that current bioequivalence guidelines for generic approval may not provide adequate assessment of the safety and efficacy of critical-dose drugs. Transplant experts at 2 recent conferences recommended more rigorous criteria for bioequivalence testing of critical-dose drugs and adoption of consistent drug substitution practices. Additional recommendations included specifying the intended formulation and instituting appropriate monitoring whenever formulations are switched. A summary of the outcomes of these conferences and practice implications for transplant coordinators is discussed.

Benefits of pre-emptive dose reduction for Sandimmune to Neoral conversion in stable renal transplant recipients.

In an effort to minimize nephrotoxicity resulting from greater exposure to cyclosporine after Sandimmune to Neoral conversion, we compared two conversion regimens using different dosing ratios. Serial serum creatinine concentrations and trough cyclosporine levels were measured in 26 patients converted from Sandimmune to Neoral using a 1:0.8 dosing ratio (Group 1) and compared to those of 26 patients converted using a 1:1 dosing ratio (Group 2). The percentage change in peak serum creatinine concentration after conversion was greater in Group 2. However, at last follow-up, the dose reductions in each group were comparable. Following conversion, patients in Group 1 required fewer dose adjustments and follow-up blood tests. Compared to conversion using a 1:1 dosing ratio, conversion from Sandimmune to Neoral using a 1:0.8 ratio results in comparable dose reductions and less short-term nephrotoxicity, while requiring less frequent laboratory monitoring.

Resolution of hepatitis B viremia in a renal transplant recipient treated with alpha-2b interferon.

A renal transplant patient developed symptomatic hepatitis after withdrawal from corticosteroids. Tests for hepatitis B e antigen and hepatitis B viral DNA were both positive prior to treatment with 1 million units alpha interferon three times weekly for 3 weeks followed by an increase to 3 million units alpha interferon three times weekly for a total of 16 weeks. At the end of treatment, hepatitis had clinically resolved with conversion to a hepatitis B e antibody positive and hepatitis B e antigen and viral DNA negative state. The renal allograft function remained excellent throughout the course of therapy with interferon.

Conversion from Sandimmune to Neoral in organ transplant recipients.

The pharmacokinetic profiles of Sandimmune and Neoral vary considerably among transplant recipients. Cyclosporine exposure is far more consistent with Neoral than it is with Sandimmune. Because intrapatient variability of drug exposure has been demonstrated to be a risk factor for chronic rejection, this difference becomes important. Neoral also has a linear dose response and a stronger correlation between trough level and drug exposure. Dose linearity greatly facilitates accurate dose titration. Results of controlled studies in which kidney, liver, and heart transplant recipients were converted from Sandimmune to Neoral have shown that conversion on a 1:1 mg basis results in more predictable bioavailability and often in reductions in cyclosporine dose. Carefully monitored conversion has not been associated with increased side effects, and any side effects that do emerge can usually be managed by taking Neoral with food, changing the dose from every 12 hours to every 8 hours, or through dose reduction.

Renal function following conversion from Sandimmune to Neoral in stable renal transplant recipients.

Conversion of stable renal transplant patients from Sandimmune to Neoral may pose a risk of short-term nephrotoxicity. Serial serum creatinine concentrations were measured in 141 kidney and simultaneous kidney-pancreas transplant recipients converted from Sandimmune to Neoral on a 1:1 dosing basis and followed for up to 11 months. Following conversion, cyclosporine dose was reduced in 74% of patients with a mean dose reduction of 22.6% for the entire cohort. Serum creatinine concentrations transiently increased to more than 30% above baseline in 48% of patients and remained more than 30% above baseline in 16% of patients. Multivariate analyses suggested that acute rejection, maintenance steroid therapy, young age, Sandimmune dose prior to conversion, and an increment in trough cyclosporine levels after conversion were positive predictors of renal insufficiency following conversion. Lower doses of Neoral should be considered at the time of conversion to minimize nephrotoxicity.

Effects of fluvastatin on hyperlipidemia after renal transplantation: influence of steroid therapy.

OBJECTIVE: To assess the efficacy and safety of fluvastatin in hypercholesterolemic, cyclosporine-treated, renal transplant recipients, and to determine whether concomitant steroid therapy in such patients alters the lipid-lowering effects of fluvastatin. DESIGN: An open-label, prospective, parallel study was performed in 20 cyclosporine-treated renal transplant recipients with hypercholesterolemia defined by a low-density lipoprotein (LDL) concentration greater than 160 mg/dL or a total cholesterol/high-density lipoprotein (HDL) concentration ratio greater than 5.0. Lipid profiles were measured before and 1 month after treatment with fluvastatin 20 mg/d. Lipid profiles in a group of patients receiving concomitant therapy with prednisone (n = 12) were compared with those of patients who had not received steroids for at least 6 months (n = 8). SETTING: The Renal Transplant Clinic at University Hospitals of Cleveland. MAIN OUTCOME MEASURES: The main outcome measures were serum concentrations of total cholesterol, LDL, HDL, and triglycerides. Treatment failure was defined by LDL concentrations persistently above 160 mg/dL after 1 month of fluvastatin therapy. Safety was assessed clinically and by serial measurements of liver enzymes and creatine phosphokinase. RESULTS: LDL concentrations decreased significantly in both the steroid-treated and steroid-free groups after 1 month of fluvastatin therapy. There was no significant change in HDL concentrations or serum triglycerides in either group. Treatment failure was more common in patients receiving steroids (4/12 patients) than in steroid-free patients (1/8 patients). After 1 month of therapy, LDL cholesterol was significantly lower in the steroid-free group (126 +/- 18 mg/dL) than in the steroid-treated group (147 +/- 23 mg/dL) (p < 0.05). There was no clinical or laboratory evidence of myonecrosis in either group. CONCLUSIONS: Low dosages of fluvastatin appear to be safe in cyclosporine-treated renal transplant recipients. Steroid-free patients exhibit a response to fluvastatin that is qualitatively similar to that of steroid-treated patients, consisting of a significant decrease in LDL concentrations and no change in HDL or serum triglyceride concentrations.

Relationship between pretransplant noncompliance and posttransplant outcomes in renal transplant recipients.

Approximately 5% to 18% of kidney transplant recipients do not comply with their posttransplant medical treatment. This study examined the relationship between pretransplant noncompliance and posttransplant outcomes. Using a longitudinal retrospective chart audit, pretransplant and posttransplant data were collected for 126 kidney transplant recipients over a 3-year period. Sixty-one percent of those identified as noncompliant before transplant lost their graft or died after transplant. Significant relationships between pretransplant noncompliance and graft loss and between pre- and posttransplant noncompliance were found. Clinicians must identify those with pretransplant noncompliance, as they are at risk for poor outcomes and might benefit from an intensive posttransplant follow-up regimen.

Determinants of long-term allograft function following steroid withdrawal in renal transplant recipients.

We retrospectively measured changes in serum creatinine concentration as estimates of changes in renal function in 96 renal transplant recipients who were withdrawn from steroid therapy, maintained on cyclosporine and azathioprine, and followed for 1 to 5 years. Multivariate analyses were used to assess the influence of cyclosporine dose and blood levels, azathioprine dose, age, sex, race, diabetes, HLA match and mismatch, PRA, and history of rejection following steroid withdrawal on long-term allograft function. Results indicate that acute rejection and cyclosporine dose are the major factors influencing long-term renal function after steroid withdrawal. In this setting, there is an inverse relationship between cyclosporine dose and serum creatinine concentration for up to 5 years. Optimal renal function is achieved in patients receiving more than 5.5 mg/kg of cyclosporine per day at the time of steroid withdrawal.

Combined kidney and pancreas transplantation.

Diabetes is the leading cause of end-stage renal disease in the United States. Combined kidney and pancreas transplantation is a safe and effective treatment option for diabetic nephropathy. During the past decade, pancreas transplants had improved outcomes as a result of improvements in pancreas recovery and preservation, the surgical procedure, immunosuppressive regimens, and immunologic monitoring. Current 1-year patient and graft survival rates are 90% and 80%, respectively, and evidence is accumulating that improvements occur in microvascular and neuropathic complications as well. Successful outcomes of kidney and pancreas transplantation are due in large part to careful nursing assessment, diagnosis, intervention, teaching, and discharge planning.