A peptide of SPARC interferes with the interaction between caspase8 and Bcl2 to resensitize chemoresistant tumors and enhance their regression in vivo

SPARC, a matricellular protein with tumor suppressor properties in certain human cancers, was initially identified in a genome-wide analysis of differentially expressed genes in chemotherapy resistance. Its exciting new role as a potential chemosensitizer arises from its ability to augment the apoptotic cascade, although the exact mechanisms are unclear. This study further examines the mechanism by which SPARC may be promoting apoptosis and identifies a smaller peptide analogue with greater chemosensitizing and tumor-regressing properties than the native protein. We examined the possibility that the apoptosis-enhancing activity of SPARC could reside within one of its three biological domains (N-terminus (NT), the follistatin-like (FS), or extracellular (EC) domains), and identified the N-terminus as the region with its chemosensitizing properties. These results were not only confirmed by studies utilizing stable cell lines overexpressing the different domains of SPARC, but as well, with a synthetic 51-aa peptide spanning the NT-domain. It revealed that the NT-domain induced a significantly greater reduction in cell viability than SPARC, and that it enhanced the apoptotic cascade via its activation of caspase 8. Moreover, in chemotherapy resistant human colon, breast and pancreatic cancer cells, its chemosensitizing properties also depended on its ability to dissociate Bcl2 from caspase 8. These observations translated to clinically significant findings in that, in-vivo, mouse tumor xenografts overexpressing the NT-domain of SPARC had significantly greater sensitivity to chemotherapy and tumor regression, even when compared to the highly-sensitive SPARC-overexpressing tumors. Our results identified an interplay between the NT-domain, Bcl2 and caspase 8 that helps augment apoptosis and as a consequence, a tumor's response to therapy. This NT-domain of SPARC and its 51-aa peptide are highly efficacious in modulating and enhancing apoptosis, thereby conferring greater chemosensitivity to resistant tumors. Our findings provide additional insight into mechanisms involved in chemotherapy resistance and a potential novel therapeutic that specifically targets this devastating phenomenon.     

Secreted protein acidic, rich in cysteine (SPARC), mediates cellular survival of gliomas through AKT activation

Secreted protein acidic, rich in cysteine (SPARC), is an extracellular matrix protein expressed in many advanced cancers, including malignant gliomas. We and others have previously shown that human glioma cell lines engineered to overexpress SPARC adopt an invasive phenotype. We now show that SPARC expression increases cell survival under stress initiated by serum withdrawal through a decrease in apoptosis. Phosphatidylinositol 3-OH kinase/AKT is a potent pro-survival pathway that contributes to the malignancy of gliomas. Cells expressing SPARC display increased AKT activation with decreased caspase 3/7 activity. Exogenous SPARC rapidly induces AKT phosphorylation, an effect that is blocked by a neutralizing SPARC antibody. Furthermore, AKT activation is essential for the anti-apoptotic effects of SPARC as the decreased apoptosis and caspase activity associated with SPARC expression can be blocked with dominant-negative AKT or a specific AKT inhibitor. As tumor cells face stressful microenvironments particularly during the process of invasion, these results suggest that SPARC functions, in part, to promote tumor progression by enabling tumor cells to survive under stressful conditions.     

Degradation of DRAK1 by CUL3/SPOP E3 Ubiquitin ligase promotes tumor growth of paclitaxel-resistant cervical cancer cells

Despite favorable responses to initial chemotherapy, drug resistance is a major cause limiting chemotherapeutic efficacy in many advanced cancers. However, mechanisms that drive drug-specific resistance in chemotherapy for patients with advanced cancers are still unclear. Here, we report a unique role of death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK1) associated with paclitaxel resistance in cervical cancer cells. Interestingly, DRAK1 protein level was markedly decreased in paclitaxel-resistant cervical cancer cells without affecting its mRNA expression, which resulted in an increase in tumor necrosis factor receptor-associated factor 6 (TRAF6) expression, as well as an activation of TRAF6-mediated nuclear factor-kappa B (NF-κB) signaling cascade, thereby promoting tumor progression. DRAK1 depletion markedly increased the chemotherapeutic IC₅₀ values of paclitaxel in cervical cancer cells. Ectopic expression of DRAK1 inhibited growth of paclitaxel-resistant cervical cancer cells in vitro and in vivo. Furthermore, DRAK1 was markedly underexpressed in chemoresistant cervical cancer patient tissues compared with chemosensitive samples. We found that DRAK1 protein was destabilized through K48-linked polyubiquitination promoted by the Cullin scaffold protein 3 (CUL3) / speckle-type POZ (poxvirus and zinc finger protein) protein (SPOP) E3 ubiquitin ligase in paclitaxel-resistant cells. Collectively, these findings suggest that DRAK1 may serve as a potential predictive biomarker for overcoming paclitaxel resistance in cervical cancer.Subject terms: Cancer therapeutic resistance, Ubiquitylation     

Association between SPARC mRNA Expression,Prognosis and Response to Neoadjuvant Chemotherapy in Early Breast Cancer: A Pooled in-silico Analysis

Introduction

SPARC is an important regulator of the extracellular matrix and has been suggested to improve delivery of albumin-bound cytotoxics. However, little is known regarding its role in breast cancer (BC).

Methods

We conducted a pooled analysis of publically available datasets, in which BC patients who received no systemic therapy or received neoadjuvant chemotherapy were eligible. Patients were assigned to molecular subtypes using PAM-50. We computed a SPARC module (SPARC7), composed of genes with an absolute correlation with SPARC >0.7. In the systemically untreated cohort, we evaluated 1) expression of SPARC/SPARC7 according to breast cancer subtype, 2) association between SPARC/SPARC7 and biological processes related to proliferation, immune and stroma, and 3) association between SPARC/SPARC7 and relapse-free survival in a Cox model in all patients and in the different molecular subtypes adjusted for tumor size, nodal status, histological grade, and age. In the neoadjuvant cohort, we evaluated the association between SPARC and pCR in a logistic regression model, adjusted for the same clinicopathologic factors.

Results

948 (10 datasets), and 791 (8 datasets) patients were included in the systemically untreated and neoadjuvant cohorts, respectively. High SPARC expression was associated with small tumor size, low histological grade and luminal-A tumors (all p<0.0001). There was a positive correlation between SPARC and stroma-related modules but negative correlation with proliferation modules. High SPARC expression was associated with poor prognosis in patients with basal and HER2+ breast cancer even after adjusting for clinicopathologic parameters. In the neoadjuvant cohort, a subgroup analysis suggested that high SPARC is associated with low rates of pCR in the HER2 subtype. Same results were observed on replacing SPARC by SPARC7.

Conclusion

This analysis suggests a potential role of SPARC in determining prognosis and response to primary chemotherapy in early BC. This information could guide further development of albumin-bound cytotoxics in BC.     

Modulation of matrix remodeling by SPARC in neoplastic progression

SPARC is a matricellular glycoprotein that mediates interactions between cells and their microenvironment. It is produced at sites of tissue remodeling, where it regulates matrix deposition and turnover, cell adhesion, and signaling by extracellular factors, exerting profound effects on tissue architecture and cell physiology. During extensive matrix remodeling in neoplastic progression, SPARC is expressed in cancer-associated stroma and in malignant cells of some types, affecting tumor development, invasion, metastases, angiogenesis and inflammation. SPARC-induced changes in the tumor microenvironment can suppress or promote progression of different cancers depending on the tissue and cell type. Understanding the mechanism of matrix remodeling and its regulation by SPARC is essential for the development of new treatment strategies for highly aggressive cancers.     

Procaspase-8失调与肿瘤细胞对TRAIL的耐受

肿瘤坏死因子相关凋亡诱导配体(TRAIL)可激活胱天蛋白酶（caspase）家族蛋白系列级联反应,最终诱导细胞凋亡. TRAIL选择性地诱导肿瘤细胞凋亡而不损伤正常细胞,使其成为治疗癌症的潜在药物靶点. 目前已知,细胞型FADD样白介素-1-β转换酶抑制蛋白(c FLIP)和凋亡抑制蛋白(IAPs)是肿瘤细胞对TRAIL耐受的主要原因.胱天蛋白酶原-8（procaspase-8）是TRAIL凋亡信号途径中的凋亡起始蛋白. 然而近年发现,在某些肿瘤细胞中procaspase-8功能失调常会阻碍凋亡信号传导,使肿瘤细胞对TRAIL诱导的凋亡产生耐受. 本文就其机制进行概述.     

Isolation and analysis of components of CD95 (APO-1/Fas) death-inducing signaling complex

CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor necrosis factor receptor superfamily. Multimerization of CD95 leads to instant recruitment of the signaling molecules FADD and caspase-8 to the activated receptor forming the death-inducing signaling complex (DISC). DISC formation is the first essential step of CD95 signaling and results in activation of caspase-8 starting a signaling cascade that leads to apoptosis. Here we describe a method for analyzing the CD95 DISC. The method is based on coimmunoprecipitation of the signaling molecules with the activated CD95 receptor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first signaling events during CD95-mediated apoptosis.     

Prostate cancer: Re-focusing on androgen receptor signaling

Prostate cancer is the most common, non-dermatologic cancer in men. Since prostate cancer is highly associated with increased age, the incidence of this disease is expected to increase as the population ages. In its initial stages prostate cancer depends upon the actions of androgen, and androgen deprivation therapy induces tumor regression. Currently, androgen deprivation is achieved by either surgical or chemical androgen blockade. Unfortunately, nearly all prostate cancer patients develop tumors that grow despite androgen blockade and ultimately relapse. Many alterations in prostate cancer cells contribute to this state. Although chemotherapy induces short remissions in some patients, there are no curative therapies for metastatic disease. This review summarizes our current understanding in androgen signaling and the mechanisms that allow tumor cells to bypass androgen manipulation therapy. The identification of novel survival pathways and effector molecules that drive androgen independent growth is necessary to develop effective therapies for advanced prostate cancers.     

Inhibition of RIP and c-FLIP enhances TRAIL-induced apoptosis in pancreatic cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it is capable of preferentially inducing apoptosis in human cancer over normal cells. The majority of human pancreatic cancers, unfortunately, are resistant to TRAIL treatment. Here, we show that the inhibition of caspase-8 cleavage is the most upstream event in TRAIL resistance in pancreatic cancers. TRAIL treatment led to the cleavage of caspase-8 and downstream caspase-9, caspase-3, and DNA fragmentation factor 45 (DFF45) in TRAIL-sensitive pancreatic cancer cell lines (BXPC-3, PACA-2). This caspase-8-initiated caspase cascade, however, was inhibited in TRAIL-resistant pancreatic cancer cell lines (PANC-1, ASPC-1, CAPAN-1, CAPAN-2). The long and short forms of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (c-FLIP(L), c-FLIP(S)) were highly expressed in the TRAIL-resistant as compared to the sensitive cells; knockdown of c-FLIP(L) and c-FLIP(S) by a short hairpin RNA (shRNA) rendered the resistant cells sensitive to TRAIL-induced apoptosis through the cleavage of caspase-8 and activation of the mitochondrial pathway. Receptor-interacting protein (RIP) has been reported in TRAIL-induced activation of NF-kappaB and we show here that knockdown of RIP sensitized the resistant cells to TRAIL-induced apoptosis. These results indicate the role of c-FLIP and RIP in caspase-8 inhibition and thus TRAIL resistance. Treatment of the resistant cells with camptothecin, celecoxib and cisplatin resulted in the downregulation of c-FLIP and caused a synergistic apoptotic effect with TRAIL. These studies therefore suggest that combination treatment with chemotherapy can overcome TRAIL resistance and enhance TRAIL therapeutic efficacy in treating pancreatic cancers.     

Hippo signaling in mammalian stem cells

Over the past decade, the Hippo signaling cascade has been linked to organ size regulation in mammals. Indeed, modulation of the Hippo pathway can have potent effects on cellular proliferation and/or apoptosis and a deregulation of the pathway often leads to tumor development. Importantly, emerging evidence indicates that the Hippo pathway can modulate its effects on tissue size by the regulation of stem and progenitor cell activity. This role has recently been associated with the central position of the pathway in sensing spatiotemporal or mechanical cues, and translating them into specific cellular outputs. These results provide an attractive model for how the Hippo cascade might sense and transduce cellular ‘neighborhood’ cues into activation of tissue-specific stem or progenitors cells. A further understanding of this process could allow the development of new therapies for various degenerative diseases and cancers. Here, we review current and emerging data linking Hippo signaling to progenitor cell function.     

Apoptosis resistance in tumor cells

Various antitumor agents induce apoptotic cell death in tumor cells. Since the apoptosis program in tumor cells plays a critical role in the chemotherapy-induced tumor cell killing, it is suggested that the defect in the signaling pathway of apoptosis could cause a new form of multidrug resistance in tumor cells. This article describes the recent findings concerning the mechanisms of chemotherapy-induced apoptosis and discusses the implication of apoptosis resistance in cancer chemotherapy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Playing the DISC: Turning on TRAIL death receptor-mediated apoptosis in cancer

Formation of the pro-apoptotic death-inducing signaling complex (DISC) can be initiated in cancer cells via binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to its two pro-apoptotic receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Primary components of the DISC are trimerized TRAIL-R1/-R2, FADD, caspase 8 and caspase 10. The anti-apoptotic protein FLIP can also be recruited to the DISC to replace caspase 8 and form an inactive complex. Caspase 8/10 processing at the DISC triggers the caspase cascade, which eventually leads to apoptotic cell death. Besides TRAIL, TRAIL-R1- or TRAIL-R2-selective variants of TRAIL and agonistic antibodies have been designed. These ligands are of interest as anti-cancer agents since they selectively kill tumor cells. To increase tumor sensitivity to TRAIL death receptor-mediated apoptosis and to overcome drug resistance, TRAIL receptor ligands have already been combined with various therapies in preclinical models. In this review, we discuss factors influencing the initial steps of the TRAIL apoptosis signaling pathway, focusing on mechanisms modulating DISC assembly and caspase activation at the DISC. These insights will direct rational design of drug combinations with TRAIL receptor ligands to maximize DISC signaling.     

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.     

Mechanisms of apoptosis modulation by curcumin: Implications for cancer therapy

Cancer incidences are growing and cause millions of deaths worldwide. Cancer therapy is one of the most important challenges in medicine. Improving therapeutic outcomes from cancer therapy is necessary for increasing patients’ survival and quality of life. Adjuvant therapy using various types of antibodies or immunomodulatory agents has suggested modulating tumor response. Resistance to apoptosis is the main reason for radioresistance and chemoresistance of most of the cancers, and also one of the pivotal targets for improving cancer therapy is the modulation of apoptosis signaling pathways. Apoptosis can be induced by intrinsic or extrinsic pathways via stimulation of several targets, such as membrane receptors of tumor necrosis factor-α and transforming growth factor-β, and also mitochondria. Curcumin is a naturally derived agent that induces apoptosis in a variety of different tumor cell lines. Curcumin also activates redox reactions within cells inducing reactive oxygen species (ROS) production that leads to the upregulation of apoptosis receptors on the tumor cell membrane. Curcumin can also upregulate the expression and activity of p53 that inhibits tumor cell proliferation and increases apoptosis. Furthermore, curcumin has a potent inhibitory effect on the activity of NF-κB and COX-2, which are involved in the overexpression of antiapoptosis genes such as Bcl-2. It can also attenuate the regulation of antiapoptosis PI3K signaling and increase the expression of MAPKs to induce endogenous production of ROS. In this paper, we aimed to review the molecular mechanisms of curcumin-induced apoptosis in cancer cells. This action of curcumin could be applicable for use as an adjuvant in combination with other modalities of cancer therapy including radiotherapy and chemotherapy.     

Androgen Receptor as a Driver of Therapeutic Resistance in Advanced Prostate Cancer

The role of the androgen receptor (AR) signaling axis in the progression of prostate cancer is a cornerstone to our understanding of the molecular mechanisms causing castration-resistant prostate cancer (CRPC). Resistance of advanced prostate cancer to available treatment options makes it a clinical challenge that results in approximately 30,000 deaths of American men every year. Since the historic discovery by Dr. Huggins more than 70 years ago, androgen deprivation therapy (ADT) has been the principal treatment for advanced prostate cancer. Initially, ADT induces apoptosis of androgen-dependent prostate cancer epithelial cells and regression of androgen-dependent tumors. However, the majority of patients with advanced prostate cancer progress and become refractory to ADT due to emergence of androgen-independent prostate cancer cells driven by aberrant AR activation. Microtubule-targeting agents such as taxanes, docetaxel and paclitaxel, have enjoyed success in the treatment of metastatic prostate cancer; although new, recently designed mitosis-specific agents, such as the polo-kinase and kinesin-inhibitors, have yielded clinically disappointing results. Docetaxel, as a first-line chemotherapy, improves prostate cancer patient survival by months, but tumor resistance to these therapeutic agents inevitably develops. On a molecular level, progression to CRPC is characterized by aberrant AR expression, de novo intraprostatic androgen production, and cross talk with other oncogenic pathways. Emerging evidence suggests that reactivation of epithelial-mesenchymal-transition (EMT) processes may facilitate the development of not only prostate cancer but also prostate cancer metastases. EMT is characterized by gain of mesenchymal characteristics and invasiveness accompanied by loss of cell polarity, with an increasing number of studies focusing on the direct involvement of androgen-AR signaling axis in EMT, tumor progression, and therapeutic resistance. In this article, we discuss the current knowledge of mechanisms via which the AR signaling drives therapeutic resistance in prostate cancer metastatic progression and the novel therapeutic interventions targeting AR in CRPC.     

Therapeutic resistance resulting from mutations in Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways

Chemotherapy remains a commonly used therapeutic approach for many cancers. Indeed chemotherapy is relatively effective for treatment of certain cancers and it may be the only therapy (besides radiotherapy) that is appropriate for certain cancers. However, a common problem with chemotherapy is the development of drug resistance. Many studies on the mechanisms of drug resistance concentrated on the expression of membrane transporters and how they could be aberrantly regulated in drug resistant cells. Attempts were made to isolate specific inhibitors which could be used to treat drug resistant patients. Unfortunately most of these drug transporter inhibitors have not proven effective for therapy. Recently the possibilities of more specific, targeted therapies have sparked the interest of clinical and basic researchers as approaches to kill cancer cells. However, there are also problems associated with these targeted therapies. Two key signaling pathways involved in the regulation of cell growth are the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. Dysregulated signaling through these pathways is often the result of genetic alterations in critical components in these pathways as well as mutations in upstream growth factor receptors. Furthermore, these pathways may be activated by chemotherapeutic drugs and ionizing radiation. This review documents how their abnormal expression can contribute to drug resistance as well as resistance to targeted therapy. This review will discuss in detail PTEN regulation as this is a critical tumor suppressor gene frequently dysregulated in human cancer which contributes to therapy resistance. Controlling the expression of these pathways could improve cancer therapy and ameliorate human health.     

The protease inhibitor bikunin,a novel anti-metastatic agent

Bikunin is a Kunitz-type protease inhibitor predominantly found in human amniotic fluid. In cancers, administration of bikunin may block tumor cell invasion by a direct inhibition of tumor cell-associated plasmin activity as well as by inhibiting urokinase-type plasminogen activator (uPA) expression at the gene and protein levels, possibly through suppression of CD44 dimerization and/or the MAP kinase signaling cascade. Treatment of cancer patients with bikunin may be beneficial in the adjuvant setting to delay the onset of metastasis development and/or in combination with cytotoxic agents to improve treatment efficacy in patients with advanced ovarian cancer.     

The epidermal growth factor receptor as a therapeutic target in epithelial ovarian cancer

A majority of patients with ovarian carcinoma who receive conventional treatment of surgical staging and platinum-based chemotherapy recur and ultimately succumb to their diseases. Novel therapies that target specific pathways involved in ovarian tumorigenesis are rapidly emerging. The epidermal growth factor receptor (EGFR) is overexpressed in 30-98% of epithelial ovarian carcinoma (EOC), and the signaling cascades activated are related with cell proliferation, migration and invasion, and angiogenesis, as well as resistance to cell apoptosis. Various trials are ongoing focusing on EGFR as an attractive target in treatment of EOC. Anti-EGFR monoclonal antibodies (MAbs), cetuximab and panitumumab, and tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, are the most advanced in clinical development. The available data suggests that MAbs and TKIs only show marginal activity when they are used alone, but combination with platinum-based chemotherapy can induce elevated overall response rate in recurrent EOC patients. Consequently, mechanisms for intrinsic and extrinsic resistance have been explored due to the poor clinical response to EGFR-targeted therapy. Careful consideration of these clinical studies and the possible mechanisms involved in resistance can provide evidence for improvements in subsequent research. Identification of responder profiles and development of rational regimen of combination therapy of EGFR-targeted therapy with other effective treatment modalities may eventually bring about substantial progress in the treatment of epithelial ovarian cancers.