Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-Specific Transcriptome and Molecular Subtype.

Longitudinal monitoring of patients with advanced cancers is crucial to evaluate both disease burden and treatment response. Current liquid biopsy approaches mostly rely on the detection of DNA-based biomarkers. However, plasma RNA analysis can unleash tremendous opportunities for tumor state interrogation and molecular subtyping. Through the application of deep learning algorithms to the deconvolved transcriptomes of RNA within plasma extracellular vesicles (evRNA), we successfully predicted consensus molecular subtypes in patients with metastatic colorectal cancer. Analysis of plasma evRNA also enabled monitoring of changes in transcriptomic subtype under treatment selection pressure and identification of molecular pathways associated with recurrence. This approach also revealed expressed gene fusions and neoepitopes from evRNA. These results demonstrate the feasibility of using transcriptomic-based liquid biopsy platforms for precision oncology approaches, spanning from the longitudinal monitoring of tumor subtype changes to the identification of expressed fusions and neoantigens as cancer-specific therapeutic targets, sans the need for tissue-based sampling. SIGNIFICANCE: The development of an approach to interrogate molecular subtypes, cancer-associated pathways, and differentially expressed genes through RNA sequencing of plasma extracellular vesicles lays the foundation for liquid biopsy-based longitudinal monitoring of patient tumor transcriptomes.

Significance of Distinct Liquid Biopsy Compartments in Evaluating Somatic Mutations for Targeted Therapy Selection in Cancer of Unknown Primary.

PURPOSE: Cancer of unknown primary (CUP) accounts for 2-5% of all cancer diagnoses, wherein standard investigations fail to reveal the original tumor site. Basket trials allocate targeted therapeutics based on actionable somatic mutations, independent of tumor entity. These trials, however, mostly rely on variants identified in tissue biopsies. Since liquid biopsies (LB) represent the overall tumor genomic landscape, they may provide an ideal diagnostic source in CUP patients. To identify the most informative liquid biopsy compartment, we compared the utility of genomic variant analysis for therapy stratification in two LB compartments (circulating cell-free (cf) and extracellular vesicle (ev) DNA). METHODS: CfDNA and evDNA from 23 CUP patients were analyzed using a targeted gene panel covering 151 genes. Identified genetic variants were interpreted regarding diagnostic and therapeutic relevance using the MetaKB knowledgebase. RESULTS: LB revealed a total of 22 somatic mutations in evDNA and/or cfDNA in 11/23 patients. Out of the 22 identified somatic variants, 14 are classified as Tier I druggable somatic variants. Comparison of variants identified in evDNA and cfDNA revealed an overlap of 58% of somatic variants in both LB compartments, whereas over 40% of variants were only found in one or the other compartment. CONCLUSION: We observed substantial overlap between somatic variants identified in evDNA and cfDNA of CUP patients. Nonetheless, interrogation of both LB compartments can potentially increase the rate of druggable alterations, stressing the significance of liquid biopsies for possible primary-independent basket and umbrella trial inclusion.

Occult polyclonality of preclinical pancreatic cancer models drives in vitro evolution.

Heterogeneity is a hallmark of cancer. The advent of single-cell technologies has helped uncover heterogeneity in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal inherent heterogeneity in assumptively monoclonal pancreatic cancer (PDAC) cell lines and patient-derived organoids (PDOs). Our findings reveal a high degree of both genomic and transcriptomic polyclonality in monolayer PDAC cell lines, custodial variation induced by growing apparently identical cell lines in different laboratories, and transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous "control" lines in experiments. We confirm these findings using a variety of independent assays, including but not limited to whole exome sequencing, single-cell copy number variation sequencing (scCNVseq), single-nuclei assay for transposase-accessible chromatin with sequencing, fluorescence in-situ hybridization, and single-cell RNA sequencing (scRNAseq). We map scRNA expression data to unique genomic clones identified by orthogonally-gathered scCNVseq data of these same PDAC cell lines. Further, while PDOs are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.

Elucidation of Tumor-Stromal Heterogeneity and the Ligand-Receptor Interactome by Single-Cell Transcriptomics in Real-world Pancreatic Cancer Biopsies.

PURPOSE: Precision medicine approaches in pancreatic ductal adenocarcinoma (PDAC) are imperative for improving disease outcomes. With molecular subtypes of PDAC gaining relevance in the context of therapeutic stratification, the ability to characterize heterogeneity of cancer-specific gene expression patterns is of great interest. In addition, understanding patterns of immune evasion within PDAC is of importance as novel immunotherapeutic strategies are developed. EXPERIMENTAL DESIGN: Single-cell RNA sequencing (scRNA-seq) is readily applicable to limited biopsies from human primary and metastatic PDAC and identifies most cancers as being an admixture of previously described epithelial transcriptomic subtypes. RESULTS: Integrative analyses of our data provide an in-depth characterization of the heterogeneity within the tumor microenvironment, including cancer-associated fibroblast subclasses, and predicts for a multitude of ligand-receptor interactions, revealing potential targets for immunotherapy approaches. CONCLUSIONS: Our analysis demonstrates that the use of de novo biopsies from patients with PDAC paired with scRNA-seq may facilitate therapeutic prediction from limited biopsy samples.

Characterisation of circulating tumour cell phenotypes identifies a partial-EMT sub-population for clinical stratification of pancreatic cancer.

BACKGROUND: Limited accessibility of the tumour precludes longitudinal characterisation for therapy guidance in pancreatic ductal adenocarcinoma (PDAC). METHODS: We utilised dielectrophoresis-field flow fractionation (DEP-FFF) to isolate circulating tumour cells (CTCs) in 272 blood draws from 74 PDAC patients (41 localised, 33 metastatic) to non-invasively monitor disease progression. RESULTS: Analysis using multiplex imaging flow cytometry revealed four distinct sub-populations of CTCs: epithelial (E-CTC), mesenchymal (M-CTC), partial epithelial-mesenchymal transition (pEMT-CTC) and stem cell-like (SC-CTC). Overall, CTC detection rate was 76.8% (209/272 draws) and total CTC counts did not correlate with any clinicopathological variables. However, the proportion of pEMT-CTCs (prop-pEMT) was correlated with advanced disease, worse progression-free and overall survival in all patients, and earlier recurrence after resection. CONCLUSION: Our results underscore the importance of immunophenotyping and quantifying specific CTC sub-populations in PDAC.

Defining the Comprehensive Genomic Landscapes of Pancreatic Ductal Adenocarcinoma Using Real-World Endoscopic Aspiration Samples.

PURPOSE: Most patients with pancreatic ductal adenocarcinoma (PDAC) present with surgically unresectable cancer. As a result, endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the most common biospecimen source available for diagnosis in treatment-naïve patients. Unfortunately, these limited samples are often not considered adequate for genomic analysis, precluding the opportunity for enrollment on precision medicine trials. EXPERIMENTAL DESIGN: Applying an epithelial cell adhesion molecule (EpCAM)-enrichment strategy, we show the feasibility of using real-world EUS-FNA for in-depth, molecular-barcoded, whole-exome sequencing (WES) and somatic copy-number alteration (SCNA) analysis in 23 patients with PDAC. RESULTS: Potentially actionable mutations were identified in >20% of patients. Further, an increased mutational burden and higher aneuploidy in WES data were associated with an adverse prognosis. To identify predictive biomarkers for first-line chemotherapy, we developed an SCNA-based complexity score that was associated with response to platinum-based regimens in this cohort. CONCLUSIONS: Collectively, these results emphasize the feasibility of real-world cytology samples for in-depth genomic characterization of PDAC and show the prognostic potential of SCNA for PDAC diagnosis.

Diagnostic value of digital droplet polymerase chain reaction and digital multiplexed detection of single-nucleotide variants in pancreatic cytology specimens collected by EUS-guided FNA.

BACKGROUND AND AIMS: EUS-guided FNA is recommended as a first-line procedure for the histopathologic diagnosis of pancreatic cancer. Molecular analysis of EUS-FNA samples might be used as an auxiliary tool to strengthen the diagnosis. The current study aimed to evaluate the diagnostic performances of K-ras testing using droplet digital polymerase chain reaction (ddPCR) and a novel single-nucleotide variant (SNV) assay performed on pancreatic EUS-FNA samples. METHODS: EUS-FNA specimens from 31 patients with pancreatic masses (22 pancreatic ductal adenocarcinomas, 7 chronic pancreatitis, and 2 pancreatic neuroendocrine tumors) were included in the study. K-ras testing was initially performed by ddPCR. In addition, mutational status was evaluated using an SNV assay by NanoString technology, using digital enumeration of unique barcoded probes to detect 97 SNVs from 24 genes of clinical significance. RESULTS: The overall specificity and sensitivity of cytologic examination were 100% and 63%, respectively. K-ras mutation testing was performed using ddPCR, and the sensitivity increased to 87% with specificity 90%. The SNV assay detected at least 1 variant in 90% of pancreatic ductal adenocarcinoma samples; the test was able to detect 2 K-ras codon 61 mutations in 2 cases of pancreatic ductal adenocarcinoma, which were missed by ddPCR. The overall diagnostic accuracy of the cytologic examination alone was 74%, and it increased to 91% when the results of both molecular tests were considered for the cases with negative and inconclusive results. CONCLUSIONS: The current study illustrated that integration of K-ras analysis with cytologic evaluation, especially in inconclusive cases, can enhance the diagnostic accuracy of EUS-FNA for pancreatic lesions.

A microfluidic device for label-free isolation of tumor cell clusters from unprocessed blood samples.

Primary cancers disseminate both single circulating tumor cells (CTCs) and CTC "clusters," the latter of which have been shown to demonstrate greater metastatic propensity and adverse impact on prognosis. Many devices developed to isolate single CTCs also capture CTC clusters, but there is translational potential for a platform specifically designed to isolate CTC clusters. Herein, we introduce our microfluidic device for isolating CTC clusters ("Microfluidic Isolation of CTC Clusters" or MICC), which is equipped with ∼10 000 trap chambers that isolate tumor cell clusters based on their large sizes and dynamic force balance against a pillar obstacle in the trap chamber. Whole blood is injected, followed by a wash step to remove blood cells and a final backflush to release intact clusters for downstream analysis. Using clusters from tumor cell-line and confocal microscopy, we verified the ability of the MICC platform to specifically capture tumor cell clusters in the trap chambers. Our flow rate optimization experiments identified 25 µl/min for blood injection, 100 µl/min as wash flow rate, and 300 µl/min as the release flow rate - indicating that 1 ml of whole blood can be processed in less than an hour. Under these optimal flow conditions, we assessed the MICC platform's capture and release performance using blood samples spiked with different concentrations of clusters, revealing a capture efficiency of 66%-87% and release efficiency of 76%-90%. The results from our study suggest that the MICC platform has the potential to isolate CTC clusters from cancer patient blood, enabling it for clinical applications in cancer management.

Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer.

BACKGROUND & AIMS: We aimed to investigate the clinical utility of circulating tumor cell DNA (ctDNA) and exosome DNA (exoDNA) in pancreatic cancer. METHODS: We collected liquid biopsy samples from 194 patients undergoing treatment for localized or metastatic pancreatic adenocarcinoma from April 7, 2015, through October 13, 2017 (425 blood samples collected before [baseline] and during therapy). Additional liquid biopsy samples were collected from 37 disease control individuals. Droplet digital polymerase chain reaction was used to determine KRAS mutant allele fraction (MAF) from ctDNA and exoDNA purified from plasma. For the longitudinal analysis, we analyzed exoDNA and ctDNA in 123 serial blood samples from 34 patients. We performed analysis including Cox regression, Fisher exact test, and Bayesian inference to associate KRAS MAFs in exoDNA and ctDNA with prognostic and predictive outcomes. RESULTS: In the 34 patients with potentially resectable tumors, an increase in exoDNA level after neoadjuvant therapy was significantly associated with disease progression (P = .003), whereas ctDNA did not show correlations with outcomes. Concordance rates of KRAS mutations present in surgically resected tissue and detected in liquid biopsy samples were greater than 95%. On univariate analysis, patients with metastases and detectable ctDNA at baseline status had significantly shorter times of progression-free survival (PFS) (hazard ratio [HR] for death, 1.8; 95% CI, 1.1-3.0; P = .019), and overall survival (OS) (HR, 2.8; 95% CI, 1.4-5.7; P = .0045) compared with patients without detectable ctDNA. On multivariate analysis, MAFs ≥5% in exoDNA were a significant predictor of PFS (HR, 2.28; 95% CI, 1.18-4.40; P = .014) and OS (HR, 3.46; 95% CI, 1.40-8.50; P = .007). A multianalyte approach showed detection of both ctDNA and exoDNA MAFs ≥5% at baseline status to be a significant predictor of OS (HR, 7.73, 95% CI, 2.61-22.91, P = .00002) on multivariate analysis. In the longitudinal analysis, an MAF peak above 1% in exoDNA was significantly associated with radiologic progression (P = .0003). CONCLUSIONS: In a prospective cohort of pancreatic cancer patients, we show how longitudinal monitoring using liquid biopsy samples through exoDNA and ctDNA provides both predictive and prognostic information relevant to therapeutic stratification.

Single-Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression.

PURPOSE: Early detection of pancreatic ductal adenocarcinoma (PDAC) remains elusive. Precursor lesions of PDAC, specifically intraductal papillary mucinous neoplasms (IPMNs), represent a bona fide pathway to invasive neoplasia, although the molecular correlates of progression remain to be fully elucidated. Single-cell transcriptomics provides a unique avenue for dissecting both the epithelial and microenvironmental heterogeneities that accompany multistep progression from noninvasive IPMNs to PDAC. EXPERIMENTAL DESIGN: Single-cell RNA sequencing was performed through droplet-based sequencing on 5,403 cells from 2 low-grade IPMNs (LGD-IPMNs), 2 high-grade IPMNs (HGD-IPMN), and 2 PDACs (all surgically resected). RESULTS: Analysis of single-cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of noninvasive dysplasia to invasive cancer. Although HGD-IPMNs expressed many core signaling pathways described in PDAC, LGD-IPMNs harbored subsets of single cells with a transcriptomic profile that overlapped with invasive cancer. Notably, a proinflammatory immune component was readily seen in low-grade IPMNs, composed of cytotoxic T cells, activated T-helper cells, and dendritic cells, which was progressively depleted during neoplastic progression, accompanied by infiltration of myeloid-derived suppressor cells. Finally, stromal myofibroblast populations were heterogeneous and acquired a previously described tumor-promoting and immune-evading phenotype during invasive carcinogenesis. CONCLUSIONS: This study demonstrates the ability to perform high-resolution profiling of the transcriptomic changes that occur during multistep progression of cystic PDAC precursors to cancer. Notably, single-cell analysis provides an unparalleled insight into both the epithelial and microenvironmental heterogeneities that accompany early cancer pathogenesis and might be a useful substrate to identify targets for cancer interception.See related commentary by Hernandez-Barco et al., p. 2027.

Dynamic changes during the treatment of pancreatic cancer.

This manuscript follows a single patient with pancreatic adenocarcinoma for a five year period, detailing the clinical record, pathology, the dynamic evolution of molecular and cellular alterations as well as the responses to treatments with chemotherapies, targeted therapies and immunotherapies. DNA and RNA samples from biopsies and blood identified a dynamic set of changes in allelic imbalances and copy number variations in response to therapies. Organoid cultures established from biopsies over time were employed for extensive drug testing to determine if this approach was feasible for treatments. When an unusual drug response was detected, an extensive RNA sequencing analysis was employed to establish novel mechanisms of action of this drug. Organoid cell cultures were employed to identify possible antigens associated with the tumor and the patient's T-cells were expanded against one of these antigens. Similar and identical T-cell receptor sequences were observed in the initial biopsy and the expanded T-cell population. Immunotherapy treatment failed to shrink the tumor, which had undergone an epithelial to mesenchymal transition prior to therapy. A warm autopsy of the metastatic lung tumor permitted an extensive analysis of tumor heterogeneity over five years of treatment and surgery. This detailed analysis of the clinical descriptions, imaging, pathology, molecular and cellular evolution of the tumors, treatments, and responses to chemotherapy, targeted therapies, and immunotherapies, as well as attempts at the development of personalized medical treatments for a single patient should provide a valuable guide to future directions in cancer treatment.

A small-molecule inhibitor of PIM kinases as a potential treatment for urothelial carcinomas.

The proto-oncogene proviral integration site for moloney murine leukemia virus (PIM) kinases (PIM-1, PIM-2, and PIM-3) are serine/threonine kinases that are involved in a number of signaling pathways important to cancer cells. PIM kinases act in downstream effector functions as inhibitors of apoptosis and as positive regulators of G1-S phase progression through the cell cycle. PIM kinases are upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, and prostate, gastric, and head and neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The aim of this investigation was to evaluate PIM expression in low- and high-grade urothelial carcinoma and to assess the role PIM function in disease progression and their potential to serve as molecular targets for therapy. One hundred thirty-seven cases of urothelial carcinoma were included in this study of surgical biopsy and resection specimens. High levels of expression of all three PIM family members were observed in both noninvasive and invasive urothelial carcinomas. The second-generation PIM inhibitor, TP-3654, displays submicromolar activity in pharmacodynamic biomarker modulation, cell proliferation studies, and colony formation assays using the UM-UC-3 bladder cancer cell line. TP-3654 displays favorable human ether-à-go-go-related gene and cytochrome P450 inhibition profiles compared with the first-generation PIM inhibitor, SGI-1776, and exhibits oral bioavailability. In vivo xenograft studies using a bladder cancer cell line show that PIM kinase inhibition can reduce tumor growth, suggesting that PIM kinase inhibitors may be active in human urothelial carcinomas.

High-throughput virtual screening identifies novel N'-(1-phenylethylidene)-benzohydrazides as potent, specific, and reversible LSD1 inhibitors.

Lysine specific demethylase 1 (LSD1) plays an important role in regulating histone lysine methylation at residues K4 and K9 on histone H3 and is an attractive therapeutic target in multiple malignancies. Here we report a structure-based virtual screen of a compound library containing ∼2 million small molecular entities. Computational docking and scoring followed by biochemical screening led to the identification of a novel N'-(1-phenylethylidene)-benzohydrazide series of LSD1 inhibitors with hits showing biochemical IC50s in the 200-400 nM range. Hit-to-lead optimization and structure-activity relationship studies aided in the discovery of compound 12, with a Ki of 31 nM. Compound 12 is reversible and specific for LSD1 as compared to the monoamine oxidases shows minimal inhibition of CYPs and hERG and inhibits proliferation and survival in several cancer cell lines, including breast and colorectal cancer. Compound 12 may be used to probe LSD1's biological role in these cancers.

Genome-wide profiling of human cap-independent translation-enhancing elements.

We report an in vitro selection strategy to identify RNA sequences that mediate cap-independent initiation of translation. This method entails mRNA display of trillions of genomic fragments, selection for initiation of translation and high-throughput deep sequencing. We identified >12,000 translation-enhancing elements (TEEs) in the human genome, generated a high-resolution map of human TEE-bearing regions (TBRs), and validated the function of a subset of sequences in vitro and in cultured cells.

Molecular Characterization of a Patient's Small Cell Carcinoma of the Ovary of the Hypercalcemic Type.

Small cell carcinoma of the ovary of the hypercalcemic type (SCCOHT) is a very rare tumor type that mainly affects young women. We report a 21-year old woman with SCCOHT. The patient initially presented with stage T3AN1MX disease and treated with surgery. The patient then received 8 cycles of multi-agent chemotherapy including cisplatin, bleomycin, cyclophosphamide, doxorubicin, and etoposide. Upon relapse, the patient underwent total abdominal hysterectomy, followed by chemotherapy with gemcitabine. The patient subsequently received radiation therapy and chemotherapy with bevacizumab, irinotecan and docetaxel. She passed away approximately 5 months after the second surgery and with her prior permission an immediate autopsy was performed. We examined the gene expression and copy number profiles of the tumor tissue samples obtained from the autopsy and compared them to normal ovary tissues. Our results indicated that although this tumor did not harbor chromosomal abnormalities nor gene copy number changes, there were significant gene expression changes in a number of genes/pathways. More than 5,000 genes showed significant differential expression in the tumor when compared to normal ovary tissue. Pathway enrichment analysis further identified several pathways/processes including the Vitamin D receptor signaling and the hedgehog signaling pathways to be significantly dysregulated. The gene expression profiling also suggests a number of agents such as pazopanib, bortezomib, 5-azacytidine, and PARP inhibitors as treatment options to possibly explore in future trials against this disease.

Validation of TPX2 as a potential therapeutic target in pancreatic cancer cells.

PURPOSE: The targeting protein for Xklp2 (TPX2) has recently gained attention as a putative oncogene possibly amplified in several human malignancies, including pancreatic adenocarcinoma. In this work, we sought to evaluate the copy number and expression of TPX2 in pancreatic cancer cell lines and tumor tissues and to further explore the potential of TPX2 as a therapeutic target. EXPERIMENTAL DESIGN: The DNA copy number and expression of the TPX2 gene were surveyed in pancreatic cancer cell lines and tumor tissues and compared with those of immortalized normal pancreatic ductal cells and normal pancreatic tissues. The cellular effects of TPX2 knockdown using small interfering RNA oligonucleotides in pancreatic cancer cells, such as growth in tissue culture, in soft agar, and in nude mice; apoptosis; and sensitivity to paclitaxel, were also investigated using various assays. RESULTS: Low-copy-number TPX2 amplification was found in pancreatic cancer cell lines and low-passage pancreatic cancer tumor xenografts. TPX2 expression was upregulated in pancreatic cancer cell lines at both the mRNA and protein levels relative to the immortalized pancreatic ductal epithelial cell line HPDE6. Immunohistochemical staining of a tissue microarray showed that TPX2 expression was higher in pancreatic tumors compared with their normal counterparts. Treatment with TPX2 targeting small interfering RNAs effectively reduced pancreatic cancer cell growth in tissue culture, induced apoptosis, and inhibited growth in soft agar and in nude mice. Knockdown of TPX2 also sensitized pancreatic cancer cells to paclitaxel treatment. CONCLUSIONS: Our results suggest that TPX2 might be an attractive target for pancreatic cancer therapy.

Identification and characterization of a novel anticancer agent with selectivity against deleted in pancreatic cancer locus 4 (DPC4)-deficient pancreatic and colon cancer cells.

OBJECTIVES: The deleted in pancreatic cancer locus 4 (DPC4)/SMAD4 tumor suppressor gene is frequently inactivated in pancreatic (approximately 55%) and colorectal cancers (approximately 30%). Like other tumor suppressor genes, the loss-of-function mutations found in the DPC4 gene are specific to cancer cells. This provides an attractive and unique opportunity for therapeutic intervention. The aim of this study was to identify and characterize small molecules that selectively kill DPC4-deficient cancer cells. METHODS: An unbiased chemical screening using isogenic cell lines that only differ in the DPC4 gene was carried out to identify positive hits. Selected hits were further verified in additional isogenic cell lines and characterized in cancer cells using several different cellular assays. RESULTS: A lead molecule, UA62784, was identified to be selectively cytotoxic against cancer cells with deficient DPC4. UA62784 preferentially induces cell cycle arrest and apoptosis in cells with deficient DPC4. It also selectively reduces the clonogenicity of DPC4-deficient cells on soft agar when compared with cells with wild type DPC4. We further demonstrate that UA62784 induces CDC2 kinase activity preferentially in DPC4-negative cells. CONCLUSIONS: UA62784 is a small molecule that selectively kills DPC4-deficient cancer cells. Its potent activity and relatively low molecular weight make it a decent candidate for further lead optimization.

Tubulin-associated proteins: Aurora and Polo-like kinases as therapeutic targets in cancer.

Tubulin is a very important target for cancer-fighting therapies; therefore, the cancer research community continues to adopt new ways of developing the therapeutic potential of tubulin and tubulin-associated proteins. Two families of tubulin-associated kinases, Aurora and Polo-like, have received significant attention regarding how they contribute to tumorigenesis and can be targeted with selective small molecule inhibitors. Aurora and Polo-like kinases play essential roles in centrosome separation, chromosome alignment and segregation, and cytokinesis. Inhibition of any of these kinases results in abnormal mitotic events (which vary depending on the particular family member) and eventually leads to apoptosis. Because of the biological consequences of inhibiting these kinases, several Aurora or Polo-like selective inhibitors have advanced to various stages of preclinical and clinical development; the most advanced are currently in phase 2 clinical trials.

Small interfering RNA-mediated knockdown of PRL phosphatases results in altered Akt phosphorylation and reduced clonogenicity of pancreatic cancer cells.

The PRL phosphatases have been implicated in cancer cell growth and metastasis in a variety of tumor types. Using cDNA microarray, we previously identified and reported PRL-1 as being highly up-regulated in pancreatic cancer cell lines. In this study, we sought to further evaluate the expression of all three PRL phosphatases in pancreatic cancer cell lines and extend our findings to in situ analysis of primary pancreatic tumors taken directly from patients. Additionally, we determine if small interfering RNA-mediated knockdown of relevant PRLs confers antitumor effects in pancreatic cancer cells. Using oligonucleotide expression arrays, mRNA levels of PRL-1 and PRL-2 but not PRL-3 were identified as up-regulated in pancreatic cancer cell lines and tumor samples taken directly from patients compared with those of normal pancreas. Focusing on PRL-1 and PRL-2, high levels of both proteins were detected in a subset of pancreatic cancer cell lines and tumor samples using Western blotting and immunohistochemistry, respectively. Small interfering RNA-mediated knockdown of PRL-1 and PRL-2 in combination resulted in a moderate reduction of cellular growth and migration in MIA PaCa-2 and PANC-1 cells. More importantly, knockdown of both PRL-1 and PRL-2 significantly inhibited colony formation of these cells in soft agar as well as serum-induced Akt phosphorylation. These data support the hypothesis that PRL phosphatases regulate key pathways involved in tumorigenesis and metastasis and that knockdown of both PRL-1 and PRL-2 is required to disrupt PRL phosphatase function in pancreatic cancer cells.

PRL phosphatases as potential molecular targets in cancer.

The phosphatase of regenerating liver (PRL) family of phosphatases, consisting of PRL-1, PRL-2, and PRL-3, represents an intriguing group of proteins being validated as biomarkers and therapeutic targets in cancer. Individual PRLs are overexpressed in a variety of cancer cell lines and tissues when compared with their normal counterparts. More importantly, several recent studies have shown that PRL-3 is expressed at higher levels and at a greater frequency in colorectal cancer metastases compared with primary colorectal tumors and normal colon tissue. Ectopic expression of PRLs in nontumorigenic cells can influence proliferation and the migratory and invasive properties of cells, while knockdown of endogenous PRL-3 or PRL-1 in cancerous cells using small interfering RNA can abrogate cell motility and ability to metastasize in a mouse model. However, the exact biological function and cellular substrates of the PRLs remain unclear. This review will discuss what is known about the PRLs, what makes the PRLs possible attractive targets for therapeutic intervention, and the possible future directions in PRL biology and inhibitor identification.