Matthew Rettig

Highly Efficient Capture of Circulating Tumor Cells by Using Nanostructured Silicon Substrates with Integrated Chaotic Micromixers

Metastases are the most common cause of cancer-related death in patients with solid tumors.[1–4] A considerable body of evidence indicates that tumor cells are shed from a primary tumor mass at the earliest stages of malignant progression[5–7]. These ‘break-away’ circulating tumor cells (CTCs)[8–11] enter the blood stream and travel to different tissues of the body, as a critical route for cancer metastasis. The current gold standard for determining tumor status requires invasive biopsy and subsequent genetic and proteomic analysis of biopsy samples. Alternatively, CTC measurement and analysis can be regarded as a “liquid biopsy” of the tumor, providing insight into tumor biology in the critical window where intervention could actually make a difference. However, detection and characterization of CTCs has been technically challenging due to their extremely low number in the bloodstream. CTCs are often found in the blood of patients with metastatic cancer (only up to hundreds of cells/mL) whereas common blood cells exist in high numbers (>109 cells/mL). Over the past decade, a diverse suite of technologies[8, 12–17] have been evolving to meet the challenge of counting and isolating CTCs from patient blood samples. Many employ different enrichment mechanisms such as immunomagnetic separation based on capture agent-labeled magnetic beads,[8, 16] microfluidics-based technologies[12, 14, 17] that enhance cell-surface contacts, and microfilter devices[13] that isolate CTCs based on size difference. The sensitivity of these emerging technologies, which is critical to their clinical utility for detecting early cancer progression (e.g., tumor invasion of vascular systems), relies on the degree of enrichment of CTCs.

AKT Activity Determines Sensitivity to Mammalian Target of Rapamycin (mTOR) Inhibitors by Regulating Cyclin D1 and c-myc Expression

Prior work demonstrates that AKT activity regulates sensitivity of cells to G1 arrest induced by mammalian target of rapamycin (mTOR) inhibitors such as rapamycin and CCI-779. To investigate this, a novel high-throughput microarray polysome analysis was performed to identify genes whose mRNA translational efficiency was differentially affected following mTOR inhibition. The analysis also allowed the assessment of steady-state transcript levels. We identified two transcripts, cyclin D1 and c-myc, which exhibited differential expression in an AKT-dependent manner: High levels of activated AKT resulted in rapamycin-induced down-regulation of expression, whereas low levels resulted in up-regulation of expression. To ectopically express these proteins we exploited the finding that the p27kip1 mRNA was efficiently translated in the face of mTOR inhibition irrespective of AKT activity. Thus, the p27kip1 5′-untranslated region was fused to the cyclin D1 and c-myc coding regions and these constructs were expressed in cells. In transfected cells, expression of cyclin D1 or c-myc was not decreased by rapamycin. Most importantly, this completely converted sensitive cells to a phenotype resistant to G1 arrest. Furthermore, the AKT-dependent differential expression patterns of these two genes was also observed in a mouse xenograft model following in vivo treatment with CCI-779. These results identify two critical downstream molecular targets whose expression is regulated by AKT activity and whose down-regulation is required for rapamycin/CCI-779 sensitivity.

Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth, metastasis and castration resistance

The transition from androgen-dependent to castration-resistant prostate cancer (CRPC) is a lethal event of uncertain molecular etiology. Comparing gene expression in isogenic androgen-dependent and CRPC xenografts, we found a reproducible increase in N-cadherin expression, which was also elevated in primary and metastatic tumors of individuals with CRPC. Ectopic expression of N-cadherin in nonmetastatic, androgen-dependent prostate cancer models caused castration resistance, invasion and metastasis. Monoclonal antibodies against the ectodomain of N-cadherin reduced proliferation, adhesion and invasion of prostate cancer cells in vitro. In vivo, these antibodies slowed the growth of multiple established CRPC xenografts, blocked local invasion and metastasis and, at higher doses, led to complete regression. N-cadherin–specific antibodies markedly delayed the time to emergence of castration resistance, markedly affected tumor histology and angiogenesis, and reduced both AKT serine-threonine kinase activity and serum interleukin-8 (IL-8) secretion. These data indicate that N-cadherin is a major cause of both prostate cancer metastasis and castration resistance. Therapeutic targeting of this factor with monoclonal antibodies may have considerable clinical benefit.

Kaposi's sarcoma-associated herpesvirus encoded vFLIP induces cellular IL-6 expression: the role of the NF-κB and JNK/AP1 pathways

The Kaposis sarcoma-associated herpesvirus (KSHV) encodes a FADD-like interferon converting enzyme or caspase 8 (FLICE) inhibitory protein (vFLIP) that prevents death receptor-mediated apoptosis by inhibiting the recruitment and activation of FLICE. Since vFLIP physically interacts with tumor necrosis factor receptor associated factor 2 (TRAF2) and TRAF2 mediates activation of the jun NH2-terminal kinase (JNK)/activation protein 1 (AP1) pathway, we hypothesized that vFLIP might also activate this pathway. To evaluate this hypothesis, we transiently and stably transfected a vFLIP expression construct and performed several complementary assays to document that vFLIP activates the JNK/AP1 pathway and does so in a TRAF-dependent fashion. As vFLIP also activates the nuclear factor κB (NF-κB) signaling pathway and the NF-κB and JNK/AP1 pathways both modulate cellular interleukin-6 (cIL-6) expression, we postulated that vFLIP induces expression of this cytokine. We show that vFLIP induces cIL-6 expression and activates the cIL-6 promoter, and maximal activation of the cIL-6 promoter by vFLIP requires NF-κB and AP1 activation. In addition, vFLIP and latency-associated nuclear antigen (LANA), another KSHV-encoded latent protein, potentiate each others ability to activate the cIL-6 promoter. Gene silencing experiments by RNA interference demonstrate that vFLIP in BCBL-1 endogenously infected primary effusion lymphoma (PEL) cells mediates JNK/AP1 activation and cIL-6 expression. Thus, we conclude that vFLIP, in addition to its known effects on NF-κB activation, also modulates the JNK/AP1 pathway and induces gene expression from the cIL-6 promoter in a JNK/AP1-dependent fashion.

High‐Purity Prostate Circulating Tumor Cell Isolation by a Polymer Nanofiber‐Embedded Microchip for Whole Exome Sequencing

Handpick single cancer cells: a modified NanoVelcro Chip is coupled with ArcturusXT laser capture microdissection (LCM) technology to enable the detection and isolation of single circulating tumor cells (CTCs) from patients with prostate cancer (PC). This new approach paves the way for conducting next-generation sequencing (NGS) on single CTCs.

Pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-κB-dependent mechanism

Constitutive nuclear factor-κB (NF-κB) activation is observed in androgen-independent prostate cancer and represents a predictor for biochemical recurrence after radical prostatectomy. Dietary agents such as pomegranate extract (PE) have received increasing attention as potential agents to prevent the onset or progression of many malignancies, including prostate cancer. Here, we show that PE inhibited NF-κB and cell viability of prostate cancer cell lines in a dose-dependent fashion in vitro. Importantly, maximal PE-induced apoptosis was dependent on PE-mediated NF-κB blockade. In the LAPC4 xenograft model, PE delayed the emergence of LAPC4 androgen-independent xenografts in castrated mice through an inhibition of proliferation and induction of apoptosis. Moreover, the observed increase in NF-κB activity during the transition from androgen dependence to androgen independence in the LAPC4 xenograft model was abrogated by PE. Our study represents the first description of PE as a promising dietary agent for the prevention of the emergence of androgen independence that is driven in part by heightened NF-κB activity. [Mol Cancer Ther 2008;7(9):2662–71]

Antitumor effects of bortezomib (PS-341) on primary effusion lymphomas

Primary effusion lymphomas (PELs) are a rare type of non-Hodgkins lymphoma that are resistant to cytotoxic chemotherapy. PELs manifest constitutive activation of nuclear factor kappa B (NF-κB), and inhibition of NF-κB induces apoptosis of PELs and sensitizes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced death. Bortezomib (PS-341), a peptidyl boronic acid inhibitor of the proteasome, is a potent agent against a wide range of hematologic malignancies and has been shown to inhibit NF-κB. Thus, we examined the cytotoxic effects of bortezomib alone and in combination with various drugs. Bortezomib potently inhibited NF-κB in PEL cells in a dose-dependent manner. In addition, bortezomib inhibited growth and induced apoptosis of PEL cell lines (IC50 values of 3.4–5.0 nM). Results of drug interactions between bortezomib and chemotherapy (doxorubicin and Taxol) were schedule-dependent: synergistic interactions were generally observed when PEL cells were pretreated with bortezomib prior to chemotherapy, whereas additive or even antagonistic interactions occurred with chemotherapy pretreatment or simultaneous treatment with bortezomib and chemotherapy. Most schedules of bortezomib and dexamethasone were synergistic, although pretreatment with dexamethasone resulted in additive interactions. Effects of combinations of bortezomib and TRAIL were generally additive. Thus, bortezomib represents a promising potential therapy for the treatment of PEL.

Inactivation of the CYLD Deubiquitinase by HPV E6 Mediates Hypoxia-Induced NF-κB Activation

The biochemical mechanisms that underlie hypoxia-induced NF-kappaB activity have remained largely undefined. Here, we find that prolonged hypoxia-induced NF-kappaB activation is restricted to cancer cell lines infected with high-risk human papillomavirus (HPV) serotypes. The HPV-encoded E6 protein is necessary and sufficient for prolonged hypoxia-induced NF-kappaB activation in these systems. The molecular target of E6 in the NF-kappaB pathway is the CYLD lysine 63 (K63) deubiquitinase, a negative regulator of the NF-kappaB pathway. Specifically, hypoxia stimulates E6-mediated ubiquitination and proteasomal degradation of CYLD. Given the established role of NF-kappaB in human carcinogenesis, these findings provide a potential molecular/viral link between hypoxia and the adverse clinical outcomes observed in HPV-associated malignancies.

NanoVelcro Chip for CTC enumeration in prostate cancer patients

Circulating tumor cells (CTCs) are one of the most crucial topics in rare cell biology and have become the focus of a significant and emerging area of cancer research. While CTC enumeration is a valid biomarker in prostate cancer, the current FDA-approved CTC technology is unable to detect CTCs in a large portion of late stage prostate cancer patients. Here we introduce the NanoVelcro CTC Chip, a device composed of a patterned silicon nanowire substrate (SiNW) and an overlaid polydimethylsiloxane (PDMS) chaotic mixer. Validated by two institutions participating in the study, the NanoVelcro Chip assay exhibits very consistent efficiency in CTC-capture from patient samples. The utilized protocol can be easily replicated at different facilities. We demonstrate the clinical utility of the NanoVelcro Chip by performing serial enumerations of CTCs in prostate cancer patients after undergoing systemic therapy. Changes in CTC numbers after 4-10 weeks of therapy were compared with their clinical responses. We observed a statistically significant reduction in CTCs counts in the clinical responders. We performed long-term follow up with serial CTC collection and enumeration in one patient observing variations in counts correlating with treatment response. This study demonstrates the consistency of the NanoVelcro Chip assay over time for CTC enumeration and also shows that continuous monitoring of CTC numbers can be employed to follow responses to different treatments and monitor disease progression.

NF-κB–Dependent Plasticity of the Epithelial to Mesenchymal Transition Induced by Von Hippel-Lindau Inactivation in Renal Cell Carcinomas

The critical downstream signaling consequences contributing to renal cancer as a result of loss of the tumor suppressor gene von Hippel-Lindau (VHL) have yet to be fully elucidated. Here, we report that VHL loss results in an epithelial to mesenchymal transition (EMT). In studies of paired isogenic cell lines, VHL silencing increased the levels of N-cadherin and vimentin and reduced the levels of E-cadherin relative to the parental VHL(+) cell line, which displayed the opposite profile. VHL(+) cells grew as clusters of cuboidal and rhomboid cells, whereas VHL-silenced cells took on an elongated, fibroblastoid morphology associated with a more highly invasive character in Matrigel chamber assays. Based on earlier evidence that VHL loss can activate NF-kappaB, a known mediator of EMT, we tested whether NF-kappaB contributed to VHL-mediated effects on EMT. On pharmacologic or molecular inhibition of NF-kappaB, VHL-silenced cells regained expression of E-cadherin, lost expression of N-cadherin, and reversed their highly invasive phenotype. Introducing a pVHL-resistant hypoxia-inducible factor 1alpha (HIF1alpha) mutant (HIFalpha(M)) into VHL(+) cells heightened NF-kappaB activity, phenocopying EMT effects produced by VHL silencing. Conversely, inhibiting the heightened NF-kappaB activity in this setting reversed the EMT phenotype. Taken together, these results suggest that VHL loss induces an EMT that is largely dependent on HIFalpha-induced NF-kappaB. Our findings rationalize targeting the NF-kappaB pathway as a therapeutic strategy to treat renal tumors characterized by biallelic VHL inactivation.