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Matthew Anaka, Omar Abdel-Rahman Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB, CanadaCorrespondence: Omar Abdel-RahmanDepartment of Oncology, Cross Cancer Institute, University of Alberta, 11560 University Avenue, Edmonton, AB, T6G 1Z2, Canada, Tel +1 780-432-8290, Fax +1 780-432-8888, Email Omar.abdelsalam@ahs.caAbstract: Fluorouracil (5FU) is the backbone chemotherapy agent in the treatment of colorectal cancer (CRC). Cardiotoxicity represents an uncommon but serious side effect of treatment with 5FU. Here, we review the current literature on 5FU-cardiotoxicity in the setting of CRC specifically, with a focus on data from the modern era of combination chemotherapy. Despite decades of study, there is little consensus on risk factors and biomarkers for 5FU-cardiotoxicity, nor how patients with CRC should be managed following a cardiotoxicity event. Given the elevated risk of recurrent cardiotoxicity on rechallenge, the use of alternative regimens that do not contain 5FU is a critical aspect of management. Data on the cardiotoxicity risk and efficacy of non-5FU regimens in CRC are therefore reviewed in detail.Keywords: fluoropyrimidine, chemotherapy, adverse events, capecitabine, fluorouracil

The DeLong Mountain Transportation System (DMTS) haul road links the Red Dog Mine-one of the world's largest zinc mines-with a shipping port on the Chukchi Sea in northwest Alaska, USA. The road traverses 32 km of National Park Service (NPS) lands managed by Cape Krusenstern National Monument (CAKR). Fugitive dusts from ore concentrate transport and mining operations have dispersed zinc (Zn), lead (Pb), cadmium (Cd), and metal sulfides onto NPS lands since the mine began operating in 1989. This study assessed the effects of metal-enriched road dusts on the diversity and community structure of lichens, bryophytes, and vascular plants in dwarf-shrub tundra within CAKR. In a Bayesian posterior predictions model, lichen species richness (LSR) was highly correlated to distance from the haul road and was distributed on the landscape consistently with the spatial patterns of Zn, Pb and Cd patterns published earlier in this journal. The mean modeled LSR of the 3000-4000 m distance class was 41.3, and LSR decreased progressively down to 9.4 species in the 0-50 m class. An ordination of 93 lichen species by 91 plots revealed strong community patterns based on distance from the haul road. The major community gradient was highly correlated (r = 0.99) with LSR and negatively correlated with Cd, Pb and Zn (-0.79 < r < -0.74). Ordinations of bryophyte classes showed less response than lichens to distance from the road and heavy metals values, and vascular plant ordination showed less still. Measures of bryophyte health such as the midrib blackening and frond width of Hylocomium splendens were positively correlated with distance from the haul road and negatively correlated with this same suite of elements. A total area of approximately 55 km2 showed moderate to strong impacts on lichens from fugitive dusts. This is equivalent to an area of almost 1 km on both sides of the haul road running 32 km through CAKR.

Spatial patterns of Zn, Pb and Cd deposition in Cape Krusenstern National Monument (CAKR), Alaska, adjacent to the Red Dog Mine haul road, were characterized in 2001 and 2006 using Hylocomium moss tissue as a biomonitor. Elevated concentrations of Cd, Pb, and Zn in moss tissue decreased logarithmically away from the haul road and the marine port. The metals concentrations in the two years were compared using Bayesian posterior predictions on a new sampling grid to which both data sets were fit. Posterior predictions were simulated 200 times both on a coarse grid of 2,357 points and by distance-based strata including subsets of these points. Compared to 2001, Zn and Pb concentrations in 2006 were 31 to 54% lower in the 3 sampling strata closest to the haul road (0-100, 100-2000 and 2000-4000 m). Pb decreased by 40% in the stratum 4,000-5,000 m from the haul road. Cd decreased significantly by 38% immediately adjacent to the road (0-100m), had an 89% probability of a small decrease 100-2000 m from the road, and showed moderate probabilities (56-71%) for increase at greater distances. There was no significant change over time (with probabilities all ≤ 85%) for any of the 3 elements in more distant reference areas (40-60 km). As in 2001, elemental concentrations in 2006 were higher on the north side of the road. Reductions in deposition have followed a large investment in infrastructure to control fugitive dust escapement at the mine and port sites, operational controls, and road dust mitigation. Fugitive dust escapement, while much reduced, is still resulting in elevated concentrations of Zn, Pb and Cd out to 5,000 m from the haul road. Zn and Pb levels were slightly above arctic baseline values in southern CAKR reference areas.

Background: Patients with advanced neuroendocrine tumors (NETs) have multiple treatment options. Ideally, treatment decisions are shared between physician and patient; however, previous studies suggest that oncologists and patients place different value on treatment attributes such as adverse event (AE) rates. High-quality information on NET patient treatment preferences may facilitate patient-centered decision making by helping clinicians understand patient priorities., Methods: This study used 2 discrete choice experiments (DCE) to elicit preferences of NET patients regarding advanced midgut and pancreatic NET (pNET) treatments. The DCEs used the "potentially all pairwise rankings of all possible alternatives" (PAPRIKA) method. The primary objective was to determine relative utility rankings for treatment attributes, including progression-free survival (PFS), treatment modality, and AE rates. Ranking of attribute profiles matching specific treatments was also determined. Levels for treatment attributes were obtained from randomized clinical trial data of NET treatments., Results: One hundred and 10 participants completed the midgut NET DCE, and 132 completed the pNET DCE. Longer PFS was the highest ranked treatment attribute in 64.5% of participants in the midgut NET DCE, and in 59% in the pNET DCE. Approximately, 40% of participants in both scenarios prioritized lower AE rates or less invasive treatment modalities over PFS. Ranking of treatment profiles in the midgut NET scenario identified 60.9% of participants favoring peptide receptor radionuclide therapy (PRRT), and 30.0% somatostatin analogue dose escalation., Conclusion: NET patients have heterogeneous priorities when choosing between treatment options based on the results of 2 independent DCEs. These results highlight the importance of shared decision making for NET patients., (© The Author(s) 2023. Published by Oxford University Press.)

Purpose: Goals of care (GoC) designations are an important part of advanced care planning (ACP) for patients with incurable cancers. Studies of outpatient oncology records show that most patients do not have GoC documented. We performed a retrospective analysis of changes in GoC designations in patients with advanced pancreatic cancer in Northern Alberta, Canada, during a system-wide ACP quality improvement initiative., Methods: Four hundred seventy-one patients with newly diagnosis of advanced, non-neuroendocrine pancreatic cancer between 2010 and 2015 in Northern Alberta, Canada, were included. The ACP initiation launched April 2014, and included educational materials for patients and families, and a coded system of GoC designations describing care philosophies and preferences for resuscitation and medical interventions. Data sources included the Alberta Cancer Registry and oncology-specific electronic medical records., Results: 25.5% of patients had a documented GoC, which increased over the study period (Mantel-Haenszel test-of-trend P < .001; increased from 7.8% in 2010 to 50.0% in 2015). GoC designations occurred later in patients who received palliative chemotherapy versus those who did not (median 130 days from diagnosis [95% CI, 76.019 to 183.981] v 36 days [95% CI, 28.107 to 43.893]; P < .001), and coincided with the end of treatment (median 4.5 days from last treatment). 64.8% of GoC designations were documented by palliative care physicians, but the proportion documented by medical oncologists increased with time (Mantel-Haenszel test-of-trend P = .020; increased from 0% in 2010 to 52.1% in 2015)., Conclusion: GoC documentation increased in the outpatient records of patients with advanced pancreatic cancer during the system-wide, multifactorial ACP initiative. GoC documentation by medical oncologists also increased. These data provide real-world evidence supporting the impact of a specific ACP initiative to improve rates of GoC designation in patients with advanced cancer., Competing Interests: Matthew AnakaStock and Other Ownership Interests: Gilead Sciences, Madrigal Pharmaceuticals, Xenon Pharma, Viking Therapeutics, Sangamo Therapeutics, Regenxbio Jennifer SpratlinHonoraria: Celgene, Sanofi, Lilly/ImClone, Eisai, Taiho PharmaceuticalConsulting or Advisory Role: Celgene, Sanofi, Lilly/ImClone, Taiho Pharmaceutical, Astellas Pharma, EisaiResearch Funding: Sanofi, Celgene (Inst)Travel, Accommodations, Expenses: Astellas PharmaNo other potential conflicts of interest were reported.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy associated with steadily increasing incidence and poor prognosis. Ongoing clinical trials are assessing the effectiveness and safety of a few immune checkpoint inhibitors (ICIs) in CCA patients. However, these ICI treatments as monotherapies may be effective for a proportion of patients with CCA. The prevalence and distribution of other immune checkpoints (ICs) in CCA remain unclear. In this pilot study, we screened databases of CCA patients for the expression of 19 ICs and assessed the prognostic significance of these ICs in CCA patients. Notably, expression of immune modulator IDO1 and PD-L1 were linked with poor overall survival, while FASLG and NT5E were related to both worse overall survival and progression-free survival. We also identified immune modulators IDO1, FASLG, CD80, HAVCR2, NT5E, CTLA-4, LGALS9, VTCN1 and TNFRSF14 that synergized with PD-L1 and correlated with worse patient outcomes. In vitro studies revealed that the expression of ICs was closely linked with aggressive CCA subpopulations, such as cancer stem cells and cells undergoing TGF-β and TNF-α-mediated epithelial-to-mesenchymal transition. These findings suggest that the aforementioned IC molecules may serve as potential prognostic biomarkers and drug targets in CCA patients, leading to lasting and durable treatment outcomes.

We propose a new process for peer review of multidisciplinary journal submissions called 'segmented peer review'. The current translational research environment increasingly requires complex and multidisciplinary studies that span multiple distinct specialties within a single paper. Such papers present logistic and practical barriers to effective peer review. To address these barriers, papers undergoing segmented peer review require authors to explicitly i) identify each of the areas of expertise required to review the paper, ii) direct each reviewer to the relevant portions of the manuscript, and iii) suggest in-field reviewers. This segmentation of the paper is then followed by a 'segmented peer review request' tailored to the expertise of each potential reviewer, with a request to confine his / her review to those 'in-scope' aspects of the paper, while de-emphasizing any optional 'out-of-scope' comments. Each reviewer indicates the fitness for publication, or suitability for revision, of their particular segment of the manuscript. The segmented peer review process is completed when the editors integrate the segmented peer reviews. We propose segmented peer review as a fit-for-purpose process with tangible advantages for authors, reviewers, and journal editors. It should reduce the specific barriers to publication inherent in the evaluation of multidisciplinary research efforts., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Elevated levels of pregnancy-associated plasma protein-A (PAPP-A) have been implicated in the pathogenesis of various malignancies, including breast cancers. Breast cancer is one of the most frequent carcinomas and is the second most common cancer type detected in women of child-bearing age. Throughout pregnancy PAPP-A is produced and secreted by the placental syncytiotrophoblast cells; co-incidentally pregnancy-associated breast cancers often have an aggressive clinical course. The components of the PAPP-A/IGF axis was assessed in a panel of breast cancer cell lines. Using neutralising antibodies the impact of PAPP-A/IGF axis on cell motility was evaluated. PAPP-A was expressed in four of the twelve breast cancer cell lines tested. Blocking PAPP-A and IGFBP4 with neutralising antibodies significantly decreased motiliy of MDA-MB-231 cells. Upregulation of PAPP-A expression in breast tumours resulted in a trend towards worse overall survival. Notably, PAPP-A expression also positively correlated with epithelial-to-mesenchymal transition markers. In conclusion, these results indicate that PAPP-A plays an important role in breast cancer progression and it may be a promising therapeutic target in breast cancer.

Sarcomas are cancers of the bone and soft tissue often defined by gene fusions. Ewing sarcoma involves fusions between EWSR1 , a gene encoding an RNA binding protein, and E26 transformation-specific (ETS) transcription factors. We explored how and when EWSR1-ETS fusions arise by studying the whole genomes of Ewing sarcomas. In 52 of 124 (42%) of tumors, the fusion gene arises by a sudden burst of complex, loop-like rearrangements, a process called chromoplexy, rather than by simple reciprocal translocations. These loops always contained the disease-defining fusion at the center, but they disrupted multiple additional genes. The loops occurred preferentially in early replicating and transcriptionally active genomic regions. Similar loops forming canonical fusions were found in three other sarcoma types. Chromoplexy-generated fusions appear to be associated with an aggressive form of Ewing sarcoma. These loops arise early, giving rise to both primary and relapse Ewing sarcoma tumors, which can continue to evolve in parallel., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3 , and VISTA were independently associated with worse prognosis, while B7-H4, CD73 , and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73 , and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB.

Purpose: Optimal first-line systemic therapy for patients with advanced nonsquamous (nonsq) non-small-cell lung cancer (NSCLC) requires confirmation of EGFR/ ALK status, which can delay treatment. We evaluated the impact of reflex testing, defined as pathologists initiating EGFR/ ALK testing at the time of diagnosis of nonsq NSCLC, on time to treatment (TTT)., Methods: We conducted a retrospective review of patients with nonsq NSCLC with medical oncology consultation at Sunnybrook Odette Cancer Centre between March 18, 2010 and April 30, 2014. Data were compared during routine and reflex testing. TTT was defined as the interval between the first medical oncology visit with advanced NSCLC and the initiation of systemic therapy., Results: A total of 306 patients were included (n = 232 for routine testing, n = 74 for reflex testing). There was a trend to improvement in median TTT with reflex testing (36 days [interquartile range {IQR}, 16 to 71 days v 26 days [IQR, 8 to 41 days], P = .071). Omitting patients with intentional delays in systemic therapy for low-volume disease, poor performance status, comorbidity management, and/or radiation therapy, median TTT improved (34 days [IQR, 15 to 67 days] v 22 days [IQR, 8 to 42 days], P = .049). Time to optimal first-line systemic therapy according to published guidelines improved (median, 36 days [IQR, 16 to 91 days] v 24 days [IQR, 8 to 43 days], P = .036). There was no impact on receipt of any first-line systemic therapy (55% v 59%, P = .66). The quality of biomarker testing improved, with fewer unsuccessful tests ( EGFR, 14% v 4%, P = .039; and ALK, 17% v 3%, P = .037)., Conclusion: Reflex testing of EGFR/ ALK improved the time to optimal systemic therapy and the quality of biomarker testing for patients with advanced nonsq NSCLC.

Background: In many cancers, microRNAs (miRs) contribute to metastatic progression by modulating phenotypic reprogramming processes such as epithelial-mesenchymal plasticity. This can be driven by miRs targeting multiple mRNA transcripts, inducing regulated changes across large sets of genes. The miR-target databases TargetScan and DIANA-microT predict putative relationships by examining sequence complementarity between miRs and mRNAs. However, it remains a challenge to identify which miR-mRNA interactions are active at endogenous expression levels, and of biological consequence., Methods: We developed a workflow to integrate TargetScan and DIANA-microT predictions into the analysis of data-driven associations calculated from transcript abundance (RNASeq) data, specifically the mutual information and Pearson's correlation metrics. We use this workflow to identify putative relationships of miR-mediated mRNA repression with strong support from both lines of evidence. Applying this approach systematically to a large, published collection of unique melanoma cell lines - the Ludwig Melbourne melanoma (LM-MEL) cell line panel - we identified putative miR-mRNA interactions that may contribute to invasiveness. This guided the selection of interactions of interest for further in vitro validation studies., Results: Several miR-mRNA regulatory relationships supported by TargetScan and DIANA-microT demonstrated differential activity across cell lines of varying matrigel invasiveness. Strong negative statistical associations for these putative regulatory relationships were consistent with target mRNA inhibition by the miR, and suggest that differential activity of such miR-mRNA relationships contribute to differences in melanoma invasiveness. Many of these relationships were reflected across the skin cutaneous melanoma TCGA dataset, indicating that these observations also show graded activity across clinical samples. Several of these miRs are implicated in cancer progression (miR-211, -340, -125b, -221, and -29b). The specific role for miR-29b-3p in melanoma has not been well studied. We experimentally validated the predicted miR-29b-3p regulation of LAMC1 and PPIC and LASP1, and show that dysregulation of miR-29b-3p or these mRNA targets can influence cellular invasiveness in vitro., Conclusions: This analytic strategy provides a comprehensive, systems-level approach to identify miR-mRNA regulation in high-throughput cancer data, identifies novel putative interactions with functional phenotypic relevance, and can be used to direct experimental resources for subsequent experimental validation. Computational scripts are available: http://github.com/uomsystemsbiology/LMMEL-miR-miner.

Background: The heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial., Methods: We iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice., Results: We demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment., Conclusion: We show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.

Epithelial-to-mesenchymal transition is a critical process that increases the malignant potential of melanoma by facilitating invasion and dissemination of tumor cells. This study identified genes involved in the regulation of cellular invasion and evaluated whether they can be targeted to inhibit melanoma invasion. We identified Peroxidasin (PXDN), Netrin 4 (NTN4) and GLIS Family Zinc Finger 3 (GLIS3) genes consistently elevated in invasive mesenchymal-like melanoma cells. These genes and proteins were highly expressed in metastatic melanoma tumors, and gene silencing led to reduced melanoma invasion in vitro. Furthermore, migration of PXDN, NTN4 or GLIS3 siRNA transfected melanoma cells was inhibited following transplantation into the embryonic chicken neural tube compared to control siRNA transfected melanoma cells. Our study suggests that PXDN, NTN4 and GLIS3 play a functional role in promoting melanoma cellular invasion, and therapeutic approaches directed toward inhibiting the action of these proteins may reduce the incidence or progression of metastasis in melanoma patients., Competing Interests: None to declare.

Background: The metabolism of cancer cells is often reprogrammed by dysregulation of metabolic enzymes. Transketolase-like 1 (TKTL1) is a homodimeric transketolase linking the pentose-phosphate pathway with the glycolytic pathway. It is generally silenced at a transcriptional level in somatic tissues. However, in human cancers its expression is associated with the acquisition of a glycolytic phenotype (the Warburg effect) by cancer cells that contributes to the progression of malignant tumors. In melanoma, defective promoter methylation results in the expression of genes and their products that can affect the tumor cell's phenotype including the modification of immune and functional characteristics. The present study evaluates the role of TKTL1 as a mediator of disease progression in melanoma associated with a defective methylation phenotype., Methods: The expression of TKTL1 in metastatic melanoma tumors and cell lines was analysed by qRT-PCR and immunohistochemistry. The promoter methylation status of TKTL1 in melanoma cells was evaluated by quantitative methylation specific PCR. Using qRT-PCR, the effect of a DNA demethylating agent 5-aza-2'-deoxycytidine (5aza) on the expression of TKTL1 was examined. Biochemical and molecular analyses such as glucose consumption, lactate production, invasion, proliferation and cell cycle progression together with ectopic expression and siRNA mediated knockdown were used to investigate the role of TKTL1 in melanoma cells., Results: Expression of TKTL1 was highly restricted in normal adult tissues and was overexpressed in a subset of metastatic melanoma tumors and derived cell lines. The TKTL1 promoter was activated by hypomethylation and treatment with 5aza induced TKTL1 expression in melanoma cells. Augmented expression of TKTL1 in melanoma cells was associated with a glycolytic phenotype. Loss and gain of function studies revealed that TKTL1 contributed to enhanced invasion of melanoma cells., Conclusions: Our data provide evidence for an important role of TKTL1 in aerobic glycolysis and tumor promotion in melanoma that may result from defective promoter methylation. This epigenetic change may enable the natural selection of tumor cells with a metabolic phenotype and thereby provide a potential therapeutic target for a subset of melanoma tumors with elevated TKTL1 expression.

Background: A current focus in cancer treatment is to broaden responses to immunotherapy. One reason these therapies may prove inadequate is that T lymphocytes fail to recognize the tumor due to differences in immunogenic epitopes presented by the cancer cells under inflammatory or non-inflammatory conditions. The antigen processing machinery of the cell, the proteasome, cleaves proteins into peptide epitopes for presentation on MHC complexes. Immunoproteasomes in inflammatory melanomas, and in antigen presenting cells of the immune system, are enzymatically different to standard proteasomes expressed by tumors with no inflammation. This corresponds to alterations in protein cleavage between proteasome subtypes, and a disparate repertoire of MHC-presented epitopes., Methods: We assessed steady state and IFNγ-induced immunoproteasome expression in melanoma cells. Using epitope specific T-lymphocyte clones, we studied processing and presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cell lines. Our experimental model allowed comparison of the processing of three distinct epitopes from a single antigen presented on the same HLA complex. We further investigated processing of these epitopes by direct inhibition, or siRNA mediated knockdown, of the immunoproteasome catalytic subunit LMP7., Results: Our data demonstrated a profound difference in the way in which immunogenic T-lymphocyte epitopes are presented by melanoma cells under IFNγ inflammatory versus non-inflammatory conditions. These alterations led to significant changes in the ability of T-lymphocytes to recognize and target melanoma cells., Conclusions: Our results illustrate a little-studied mechanism of immune escape by tumor cells which, with appropriate understanding and treatment, may be reversible. These data have implications for the design of cancer vaccines and adoptive T cell therapies.

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

Melanoma is the most common cancer diagnosed in pregnant women and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. Here, we report that melanoma migration, invasiveness and progression are promoted by Pregnancy-Associated Plasma Protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of IGF1 by cleaving it from a circulating complex formed with IGFBP4. We show that PAPPA is widely expressed by metastatic melanoma tumors and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in vivo within the embryonic chicken neural tube. PAPPA-enriched pregnancy serum treatment enhanced melanoma motility in vitro. Furthermore, we report that IGF1 can induce the phenotypic and functional effects of epithelial-to-mesenchymal transition (EMT) in melanoma cells. In this study, we establish a clear relationship between a pregnancy-associated protein PAPPA, melanoma and functional effects mediated through IGF1 that provides a plausible mechanism for accelerated melanoma progression during pregnancy. This opens the possibility of targeting the PAPPA/IGF1 axis therapeutically.

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conflicting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro, and reduced xenograft growth in vivo. FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma.

Background: Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma., Methods: Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays., Results: MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse., Conclusion: Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

The ability of cell lines to accurately represent cancer is a major concern in preclinical research. Culture of glioma cells as neurospheres in stem cell media (SCM) has been shown to better represent the genotype and phenotype of primary glioblastoma in comparison to serum cell lines. Despite the use of neurosphere-like models of many malignancies, there has been no robust analysis of whether other cancers benefit from a more representative phenotype and genotype when cultured in SCM. We analyzed the growth properties, transcriptional profile, and genotype of melanoma cells grown de novo in SCM, as while melanocytes share a common precursor with neural cells, melanoma frequently demonstrates divergent behavior in cancer stem cell assays. SCM culture of melanoma cells induced a neural lineage gene expression profile that was not representative of matched patient tissue samples and which could be induced in serum cell lines by switching them into SCM. There was no enrichment for expression of putative melanoma stem cell markers, but the SCM expression profile did overlap significantly with that of SCM cultures of glioma, suggesting that the observed phenotype is media-specific rather than melanoma-specific. Xenografts derived from either culture condition provided the best representation of melanoma in situ. Finally, SCM culture of melanoma did not prevent ongoing acquisition of DNA copy number abnormalities. In conclusion, SCM culture of melanoma does not provide a better representation of the phenotype or genotype of metastatic melanoma, and the resulting neural bias could potentially confound therapeutic target identification., (Copyright © 2011 AlphaMed Press.)

Genomic imprinting is a process that marks DNA, causing a change in gene or chromosome behavior, depending on the sex of the transmitting parent. In mammals, most examples of genomic imprinting affect the transcription of individual or small clusters of genes whereas in insects, genomic imprinting tends to silence entire chromosomes. This has been interpreted as evidence of independent evolutionary origins for imprinting. To investigate how these types of imprinting are related, we performed a phenotypic, molecular, and cytological analysis of an imprinted chromosome in Drosophila melanogaster. Analysis of this chromosome reveals that the imprint results in transcriptional silencing. Yet, the domain of transcriptional silencing is very large, extending at least 1.2 Mb and encompassing over 100 genes, and is associated with decreased somatic polytenization of the entire chromosome. We propose that repression of somatic replication in polytenized cells, as a secondary response to the imprint, acts to extend the size of the imprinted domain to an entire chromosome. Thus, imprinting in Drosophila has properties of both typical mammalian and insect imprinting which suggests that genomic imprinting in Drosophila and mammals is not fundamentally different; imprinting is manifest as transcriptional silencing of a few genes or silencing of an entire chromosome depending on secondary processes such as differences in gene density and polytenization.

The white gene of Drosophila melanogaster has been extensively studied, yet it is still not understood how its ectopic overexpression induces male-male courtship. To investigate the cellular basis of this behavior, we examined the sexual behavior of several classes of mutants. We find that male-male courtship is seen not only in flies overexpressing the white gene, but also in mutants expected to have mislocalized White protein. This finding confirms that mislocalizing White transporter in the cells in which it is normally expressed will produce male-male courtship behaviors; the courtship behavior is not an indirect consequence of aberrant physiological changes elsewhere in the body. Male-male courtship is also seen in some mutants with altered monoamine metabolism and deficits in learning and memory, but can be distinguished from that produced by White mislocalization by its reduced intensity and locomotor activity. Double mutants overexpressing white and with mutations in genes for serotonergic neurons suggest that male-male courtship produced by mislocalizing White may not be mediated exclusively by serotonergic neurons. We also find decreased olfactory learning in white mutants and in individuals with mutations in the genes for White's binding partners, brown and scarlet. Finally, in cultured Drosophila and mammalian cells, the White transporter is found in the endosomal compartment. The additional genes identified here as being involved in male-male courtship increase the repertoire of mutations available to study sexual behavior in Drosophila.