Prostate cancer growth depends on androgen receptor (AR) signaling. Androgen ablation therapy induces expression of constitutively active AR splice variants which drive disease progression. Taxanes are a standard of care therapy in castration-resistant prostate cancer (CRPC), however, mechanisms underlying the clinical activity of taxanes are poorly understood. Recent work suggests that the microtubule network of prostate cells is critical for AR nuclear translocation and activity. In this study, we employed a set of AR deletion mutants to identify the microtubule-binding domain of AR, which encompasses the DNA binding domain plus hinge region. We report that two clinically relevant AR splice variants, ARv567 and ARv7, differentially associate with microtubules and dynein motor protein, thereby resulting in differential taxane sensitivity in vitro and in vivo. ARv7, which lacks the hinge region, did not co-sediment with microtubules or co-precipitate with dynein motor protein, unlike ARv567. Mechanistic investigations revealed that the nuclear accumulation and transcriptional activity of ARv7 was unaffected by taxane treatment. In contrast, the microtubule-interacting splice variant ARv567 was sensitive to taxane-induced microtubule stabilization. In ARv567-expressing LuCap86.2 tumor xenografts, docetaxel treatment was highly efficacious, whereas ARv7-expressing LuCap23.1 tumors xenografts displayed docetaxel resistance. Our results suggest that AR variants which accumulate in CRPC cells utilize distinct pathways of nuclear import that affect the antitumor efficacy of taxanes, suggesting a mechanistic rationale to customize treatments for CRPC patients which might improve outcomes.

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC 80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non-cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab-maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADCresistant cell models retain sensitivity to other ADCs or standardof-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2þ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 þ tumors, are not well understood. We used HER2 þ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an "onoff" routine until a T-DM1-resistant population was generated. T-DM1-resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavablelinked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells.

Drug resistance limits the effectiveness of cancer therapies. Despite attempts to develop curative anticancer treatments, tumors evolve evasive mechanisms limiting durable responses. Hence, diverse therapies are used to attack cancer, including cytotoxic and targeted agents. Antibody-drug conjugates (ADC) are biotherapeutics designed to deliver potent cytotoxins to cancer cells via tumor-specific antigens. Little is known about the clinical manifestations of drug resistance to this class of therapy; however, recent preclinical studies reveal potential mechanisms of resistance. Because ADCs are a combination of antibody and small molecule cytotoxin, multifactorial modes of resistance are emerging that are inherent to the structure and function of the ADC. Decreased cell-surface antigen reduces antibody binding, whereas elevated drug transporters such as MDR1 and MRP1 reduce effectiveness of the payload. Inherent to the uniqueness of the ADC, other novel resistance mechanisms are emerging, including altered antibody trafficking, ADC processing, and intracellular drug release. Most importantly, the modular nature of the ADC allows components to be switched and replaced, enabling development of second-generation ADCs that overcome acquired resistance. This review is intended to highlight recent progress in our understanding of ADC resistance, including approaches to create preclinical ADC-refractory models and to characterize their emerging mechanisms of resistance. Mol Cancer Ther; 15(12); 2825-34. ©2016 AACR.

Taxanes are the only chemotherapies used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study, we show that ERG over-expression in in vitro and in vivo models of CRPC is associated with decreased sensitivity to taxanes. ERG affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of docetaxel or cabazitaxel with tubulin. Finally, analysis of a cohort of 34 men with metastatic CRPC treated with docetaxel chemotherapy reveals that ERG-overexpressing prostate cancers have twice the chance of docetaxel resistance than ERG-negative cancers. Our data suggest that ERG plays a role beyond regulating gene expression and functions outside the nucleus to cooperate with tubulin towards taxane in sensitivity. Determining ERG rearrangement status may aid in patient selection for docetaxel or cabazitaxel therapy and/or influence co-targeting approaches.

Circulating Tumor Cells (CTCs) have emerged as a reliable source of tumor cells, and their concentration has prognostic implications. CTC capture offers real-time access to cancer tissue without the need of an invasive biopsy, while their phenotypic and molecular interrogation can provide insight into the biological changes of the tumor that occur during treatment. The majority of the CTC capture methods are based on EpCAM expression as surface marker of tumor-derived cells. However, EpCAM protein expression levels can be significantly down regulated during cancer progression as consequence of the process of epithelial to mesenchymal transition. In this paper, we describe a novel HER2 (Human Epidermal Receptor 2)-based microfluidic device for the isolation of CTCs from peripheral blood of patients with HER2-expressing solid tumors. We selected HER2 as an alternative to EpCAM, as the receptor is biologically and therapeutically relevant in several solid tumors, like breast cancer (BC), where it is overexpressed in 30% of the patients and expressed in 90%, and gastric cancer (GC), in which HER2 presence is identified in more than 60% of the cases. We tested the performance of various anti HER2 antibodies in a panel of nine different BC cell lines with varying HER2 protein expression levels, using immunoblotting, confocal microscopy, live cells imaging and flow cytometry analyses. The antibody associated with the highest capture efficiency and sensitivity for HER2 expressing cells on the microfluidic device, was the one that performed best in live cells imaging and flow cytometry assays as opposed to the fixed cell analyses, suggesting that recognition of the native conformation of HER2 extracellular epitope on living cells was essential for specificity and sensitivity of CTC capture. Next, we tested the performance of the HER2 microfluidic device using blood from metastatic breast and gastric cancer patients. The HER2 microfluidic device exhibited CTC capture in 9/9 blood samples. Thus, the described HER2-based microfluidic device can be considered as a valid clinically relevant method for CTC capture in HER2 expressing solid cancers.

The taxanes are effective microtubule-stabilizing chemotherapy drugs used in the treatment of various solid tumors. However, the emergence of drug resistance hampers their clinical efficacy. The molecular basis of clinical taxane resistance remains poorly understood. Breast cancer 1, early onset gene, BRCA1, is a tumor-suppressor gene, whose expression has been correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer. However, the molecular mechanism underlying the relationship between BRCA1 (B1) expression and taxane activity remains unclear. To this end, we created a stable B1 knockdown A549 cell line (B1-KD) to investigate B1’s role in microtubule biology and response to taxane treatment. We show that B1-KD rendered A549 cells resistant to paclitaxel (PTX), phenocopying clinical studies showing that low B1 expression correlated with taxane resistance. As previously reported, we show that loss of B1 enhanced centrosomal γ-tubulin localization and microtubule nucleation. Interestingly, we found that the B1-KD cells exhibited increased microtubule dynamics as compared with parental A549 cells, as assessed by live-cell confocal microscopy using enhanced green fluorescent protein-tagged α-tubulin or EB1 protein. In addition, we showed that loss of B1 impairs the ability of PTX to induce microtubule polymerization using immunofluorescence microscopy and a cell-based tubulin polymerization assay. Furthermore, B1-KD cells exhibited significantly lower intracellular binding of a fluorescently labeled PTX to microtubules. Recent studies have shown that PTX-stabilized microtubules serves as a scaffold for pro-caspase-8 binding and induction of apoptosis downstream of induced-proximity activation of caspase-8. Here we show that loss of B1 reduces the association of pro-caspase-8 with microtubules and subsequently leads to impaired PTX-induced activation of apoptosis. Taken together, our data show that B1 regulates indirectly endogenous microtubule dynamics and stability while its loss leads to microtubules that are more dynamic and less susceptible to PTX-induced stabilization conferring taxane resistance.

Nicotinamide adenine dinucleotide (NAD + ) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD + -regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD + . Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD + levels. We find that these effects of NAD + are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD + on microtubule polymers. Taken together, these data demonstrate that NAD + and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.N icotinamide adenine dinucleotide (NAD + ) is an endogenous dinucleotide that is present in the cytosol, nucleus, and mitochondria. Athough it serves an important role as a redox cofactor in metabolism, NAD + is also a substrate for several families of enzymes, including the poly(ADP ribose) polymerases and the sirtuin deacetylase enzymes (reviewed in refs. 1 and 2). The level of intracellular NAD + is regulated by many factors, including diet and energy status (3), axonal injury (4), DNA damage (5), and certain disease states (6), suggesting that NAD + -dependent signaling is dynamically modulated in diverse contexts.NAD + -dependent signaling can be induced by treatment of cells with exogenous NAD + , which increases intracellular NAD + levels and results in diverse effects in cells and animals. These effects include enhanced oxygen consumption and ATP production (7), as well as protection from genotoxic stress and apoptosis (3). Mice treated with nicotinamide riboside, a NAD + precursor that is metabolized into NAD + , have enhanced oxidative metabolism, increased insulin sensitivity, and protection from high-fat diet-induced obesity (8). These results demonstrate that NAD + -dependent pathways can enhance metabolic function and improve a variety of disease phenotypes.An NAD + -regulated pathway also inhibits axonal degeneration elicited by axonal transection (4). Treatment of axons with 5-20 mM NAD + markedly delays the axon degenerative process (9). Additionally, animals that express the Wallerian degeneration slow (Wld S ) protein, a fusion of the NAD + biosynthetic enzyme Nicotinamide mononucleotide adenylyl transferase 1 and Ube4a, exhibit markedly delayed degeneration of the distal axonal fragment after axonal transection (10), and expression of Wld S mitigates disease phenotypes in several neurodegenerative disease models (11)(12)(13)(14). Thus, understanding the intracellular pathways regulated by NAD + may be important for understanding the pa...