Preclinical Evaluation of 68Ga-Labeled CSPG4-Targeting Peptides for PET/CT Imaging in Pancreatic and Gastric Carcinoma.

The proteoglycan CSPG4 [chondroitin sulfate proteoglycan 4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP) or neuron-glial antigen 2 (NG2)] has been associated with progression of melanoma, and intervention to reduce CSPG4 has been considered as treatment of melanoma. In recent work, we have reported that decline in the enzyme arylsulfatase B (ARSB, N-acetylgalactosamine 4-sulfatase) is associated with malignancy, including prostate, mammary, and colon. ARSB is the enzyme that removes 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate (DS), and thereby regulates the degradation of the sulfated glycosaminoglycan (GAG) chain. Hence, the absence of ARSB leads to the accumulation of more highly sulfated C4S or DS. In prior published work, decline in ARSB and the associated increase in C4S led to transcriptional events, mediated through a decline in galectin-3 binding to the more highly sulfated C4S present. Transcription factors activator protein 1 (AP-1) and Specificity protein 1 (Sp1) interacted with galectin-3 to mediate the increased transcription of versican and Wnt9A. In this study, the mechanism by which decline in ARSB increased expression of CSPG4 and. increased the invasiveness of melanoma cell lines was addressed. Techniques included: cell culture of normal melanocytes (ATCC PCS 200-013) and melanoma cell lines obtained from radial or vertical growth phase [WM1552C, WM1552C/mock (mock CSPG4 transfection), WM1552C/MCSP (CSPG4 transfected), WM1552C/MCSPÄCD (transfected with CSPG4 without the cytoplasmic domain), WM35] and from metastatic melanoma (1205 Lu); QPCR of CSPG4; ARSB activity assay using the exogenous substrate 4-methylumbelliferylsulfate; silencing of ARSB by siRNA; invasion assay; measurements of total sulfated glycosaminoglycans and C4S using 1,9-dimethylmethylene blue dye; and measurements of matrix metalloproteinase (MMP) activity. The results show that ARSB activity was reduced in the metastatic melanoma cells to 51% of the level in the normal melanocytes. Silencing of ARSB was associated with increase to 2.8 times the baseline level in the mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4) in the normal melanocytes. Invasiveness of the melanoma cells increased by 70% when ARSB was silenced, with corresponding increase in MMP activity. Since CSPG4 has C4S attachments, the effects of ARSB on CSPG4 expression may affect the availability of C4S GAG chains to interact with extracellular matrix components and affect invasiveness. The findings indicate that decline in ARSB and the associated increase in C4S have profound effects on the invasive potential of melanoma and that increased attention to ARSB and C4S may yield new therapeutic interventions. Citation Format: Sumit Bhattacharyya, Leo Feferman, Kaoru Terai, Arkadiusz A. Dudek, Joanne K. Tobacman. Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 697.

Simple SummaryMany solid tumours, such as those of the breast, colon, and prostate, have well established molecular markers of malignancy. However, in certain cancers, such as squamous cell carcinoma (SCC), few clinically useful biomarkers exist. Recently, several candidates that might be used for diagnosis in SCC have been proposed. The purpose of this review is to discuss chondroitin sulfate (CS) proteoglycan 4 (CSPG4) as a tumour biomarker and explain why its expression might be considered in clinical decision making for patients with SCCs, including those of the head and neck, and those arising from rare genetic disorders, such as epidermolysis bullosa (EB).Chondroitin sulfate (CS) proteoglycan 4 (CSPG4) is a cell surface proteoglycan that is currently under investigation as a marker of cancer malignancy, and as a potential target of anticancer drug treatment. CSPG4 acts as a driver of tumourigenesis by regulating turnover of the extracellular matrix (ECM) to promote tumour cell invasion, migration as well as inflammation and angiogenesis. While CSPG4 has been widely studied in certain malignancies, such as melanoma, evidence is emerging from global gene expression studies, which suggests a role for CSPG4 in squamous cell carcinoma (SCC). While relatively treatable, lack of widely agreed upon diagnostic markers for SCCs is problematic, especially for clinicians managing certain patients, including those who are aged or infirm, as well as those with underlying conditions such as epidermolysis bullosa (EB), for which a delayed diagnosis is likely lethal. In this review, we have discussed the structure of CSPG4, and quantitatively analysed CSPG4 expression in the tissues and pathologies where it has been identified to determine the usefulness of CSPG4 expression as a diagnostic marker and therapeutic target in management of malignant SCC.

Abstract #5046 Introduction: Basal-like breast cancer is associated with high grade, poor prognosis, and younger patient age. Because tumors are estrogen receptor (ER) and HER2 negative, effective targeted treatment for basal breast cancer remains elusive. The lack of efficacy of currently used chemotherapies may reflect their inability to eradicate cancer stem cells (CSC) within a basal breast tumor.
 Chondroitin sulfate proteoglycan 4 (CSPG4), a molecule initially identified in melanoma cells, is a cell surface antigen involved in migration and invasion of tumor cells. The rat homolog of the human CSPG4, named NG2, is expressed by progenitor cells in several types of tissues. Furthermore, NG2 expression is typically maximal when progenitor cells are mitotic and motile. As one important characteristic of cancer stem cells is their ability to migrate and metastasize, and both CSPG4 and NG2 play a role in tumor cell migration, we tested whether CSPG4 is expressed on cells with the CSC phenotype in ER negative/HER2 negative (basal-like) breast cancer, and can thus serve as a target for immunotherapy of breast CSC.
 Methods and Results: The mRNA levels of CSPG4 in ER- and Her2- breast cancer were compared to ER-/Her2+, ER+/Her2-, and ER+/Her2+ breast cancer using a publicly available, clinically annotated breast cancer data set. The gene expression level of CSPG4 was elevated in the ER- and Her2- subgroup when compared to ER-/Her2+ (Mann Whitney, p=0.03), ER+/Her2- (Mann Whitney, p=0.01), and ER+/Her2+ (Mann Whitney, p=0.06). These results suggest that CSPG4 expression is best correlated with the basal phenotype of breast cancer. Flow cytometric analysis showed that CSPG4 is expressed on CD44+, CD24-/lo cells in the human basal breast cancer cell lines, i.e., MDA-MD-231, MDA-MB-435, HS578T and SUM149, but is not detectable on the luminal breast cancer cell lines, i.e., MCF-7, SK-BR-3 and T-47D. Furthermore CSPG4 is expressed on the cell surface of putative breast CSC identified as lineage-negative (CD2,3,10,16,18,31,45,64,140b), CD44+, CD24-/lo cells in pleural effusions of 12 patients with breast cancer. Further, inoculation of flow cytometric sorted CD44+CD24-/low CSPG4+ cells from the MDA-MB-231-luciferease expressing cell line, to the mammary fat pads of immunodeficient mice, led to the development of tumors detected by whole-body bioluminescent reporter imaging 3 weeks after the injection. In contrast, inoculation of CD44+CD24-/low CSPG4- cells, sorted from the same cell line, did not generate tumors. More importantly, CSPG4-specific mAb significantly inhibited post-surgery tumor recurrence and lung metastases in SCID mice transplanted with the human basal breast cancer cell line MDA-MB-435, which has the putative breast CSC phenotype (CD44+, CD24-/lo) in more than 99% of cells.
 Conclusion: These results suggest that the CSPG4 is a potential new immunotherapeutic target for basal-like breast cancer and breast CSC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5046.

Background: Chondroitin sulfate proteoglycan 4 (CSPG4) was first identified as a melanoma specific antigen and demonstrated to stabilize cell-substratum interactions during early events of tumor cell spreading on endothelial cells and matrix proteins. In invasive breast cancers (IBCs), CSPG4 has been reported to be highly expressed in the triple-negative (TN) subtype. Western blotting analyses suggest that CSPG4 is expressed on TN IBC lines. Here we further explored the specific gene expression (GE) profiles associated with CSPG4 using the available IBC cases from The Cancer Genome Atlas (TCGA) project towards identification of potential drug targets for patients with TN IBC. Methods: The GE data (Agilent, log2 transformed) of 459 IBC tumors were downloaded from the TCGA data portal. PAM50 classification results of all samples were obtained from the TCGA breast cancer AWG group, including 203 Luminal A (LA), 113 Luminal B (LB), 51 HER2+, 84 basal-like (Basal), and 8 Normal-like (not used). Clinicopathologic data (age, race, T-, N-, and M-stages) were also obtained from the AWG group. Non-parametric Kruskal-Wallis test was used for cross-subtype analysis of CSPG4 expression, followed by Wilcoxon-Mann-Whitney test for pairwise comparison with Bonferroni adjustment. Chi-square tests were performed for categorical variation analyses. Differentially Expressed Gene (DEG) analysis was performed using the Bioconductor “samr” package for two class unpaired comparison, with p < 0.05 and FDR < 0.10 as the cut-off for DEGs. All other statistical analyses were performed using SAS. Results: GE level of CSPG4 was highest in the Basal subtypes (mean±SD, −0.92±1.37) compared to all other subtypes of IBCs, including LA (−1.50±0.66, p = 0.0013), LB (−1.86±0.68, p < 0.0001), and HER2 (−1.69±0.60, p = 0.0014). GE in LA was also significantly higher compared to LB (p < 0.0001). The Basal subtype exhibited a much wider variation of the expression level compared to other subtypes. To better understand this variation, we analyzed the age, T, N, and M across the 4 quartiles of the CSPG4 GE levels in the Basal subtype. Only N was significantly associated with the quartiles (p = 0.04), with N-positive rates of 45.8%, 54.2%, 32.0%, and 16.7% from the lowest to the highest quartiles respectively. Further DEG analysis between the top and bottom quartiles of the samples identified 46 genes, including MIA, SOX10, MSMB etc. Many of these genes showed a strikingly similar expression distribution pattern to CSPG4. Discussion: Our study confirmed the higher GE of CSPG4 in Basal subtype. In this subtype, patients expressing high CSPG4 in the tumor show a lower rate of local lymph node metastasis, implying that these tumors may not metastasize via the lymphatic vascular system. A number of DEGs were identified that are positively associated with CSPG4 expression. Although the roles these molecules play in promoting IBC progression needs to be further evaluated, our study may lead to the identification of a drug target for the Basal subtype of IBC patients. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-06.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin-blistering disorder that often progresses to metastatic cutaneous squamous cell carcinoma (cSCC) at chronic wound sites. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell-surface proteoglycan that is an oncoantigen in multiple malignancies, where it modulates oncogenic signalling, drives epithelial-to-mesenchymal transition (EMT) and enables cell motility. To evaluate CSPG4 expression and function in RDEB cSCC. RDEB cSCC cell lines were used to assess CSPG4-dependent changes in invasive potential, transforming growth factor (TGF)-β1-stimulated signal activation and clinically relevant cytopathology metrics in an in vitro full-thickness tumour model. CSPG4 expression in RDEB cSCC and non-RDEB cSCC tumours was analysed via immunohistochemistry and single-cell RNA sequencing (scRNA-Seq), respectively. Inhibiting CSPG4 expression reduced invasive potential in multiple RDEB cSCC cell lines and altered membrane-proximal TGF-β signal activation via changes in SMAD3 phosphorylation. CSPG4 expression was uniformly localized to basal layer keratinocytes in fibrotic RDEB skin and tumour cells at the tumour-stroma interface at the invasive front in RDEB cSCC tumours in vivo. Analysis of published scRNA-Seq data revealed that CSPG4 expression was correlated with an enhanced EMT transcriptomic signature in cells at the tumour-stroma interface of non-RDEB cSCC tumours. Cytopathological metrics, for example nucleus : cell area ratio, were influenced by CSPG4 expression in in vitro tumour models. We determined that CSPG4 expression in RDEB cSCC cell lines enhanced the invasive potential of tumours. Mechanistically, CSPG4 was found to enhance membrane-proximal TGF-β-stimulated signalling via SMAD3, which is a key mediator of EMT in RDEB cSCC. The implication of these studies is that CSPG4 may represent a therapeutic target that can be leveraged for the clinical management of patients with RDEB cSCC.

The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function

Chimeric antigen receptor T (CAR-T) cell therapy is under clinical investigation in patients with metastatic triple-negative breast cancer (TNBC). However, the identification of targetable antigens remains a high priority to avoid toxicity and prevent tumor escape. Here we analyzed the gene expression of B7-H3 (CD276) and chondroitin sulfate proteoglycan 4 (CSPG4) in 98 TNBC samples identified in the AURORA US Network and Rapid Autopsy RNA sequencing data set at University of North Carolina (UNC). We then performed immunohistochemistry analysis for B7-H3 and CSPG4 protein expression in 151 TNBC samples collected at UNC. Finally, the validity of the proposed B7-H3 and CSGP4 co-targeting was tested in clinically relevant TNBC patient derived xenograft (PDX) models. We observed that CD276 and CSPG4 genes are broadly and comparably expressed in TNBC samples, and gene expression is generally conserved in tumor metastases. None of the TNBC analyzed met the criteria for simultaneous low expression of CSPG4 and CD276 genes. Immunohistochemistry analysis showed a median H-score of 138 (105-168, lower and upper quartile, respectively) for B7-H3 expression and a median H-score of 33 (14-78 lower and upper quartile, respectively) for CSPG4 expression. Notably, 49% of the TNBC cores with B7-H3 H-score ≤105 exhibited a CSPG4 H-score exceeding its median value, and 37% and 18% of the TNBC cores with low B7-H3 expression scored CSPG4 expression above its median H-score or exceeded its upper quartile, respectively, confirming that at least one of these two proteins is expressed in 94% of the analyzed tumors. Finally, optimized dual-specific B7-H3 and CSPG4 CAR-T cells eradicated tumors with mixed antigen expression in TNBC PDX models. These data highlight the clinical potential of the proposed approach that could be applicable to the great majority of patients with TNBC as well as most of patients with breast cancer in general.

Preeclampsia (PE) is a condition of pregnancy in which symptoms of hypertension develop after 20weeks of gestation. it can lead to placental dysfunction, maternal and perinatal mortality and morbidity. The incidence of PE is increasing, posing a serious threat to the lives of pregnant women and their unborn children. Currently, most of the research on the pathogenesis of PE has focused on placenta, However, maternal decidualization is the basis for placental formation and growth. Chondroitin sulfate proteoglycan 4 (CSPG4) is a transmembrane protein that plays a role in cell proliferation, invasion, and migration. However, its function during decidualization is not yet understood. In this study, we investigated the role of CSPG4 and found that its expression was significantly down-regulated in the decidual tissue of patients with severe PE compared to normal pregnant women. During artificially induced decidualization, CSPG4 expression was significantly increased. Knockdown of CSPG4 by small interfering RNA inhibited decidualization, which, in turn, inhibited the invasion of trophoblast cells. In both pseudopregnant and pregnant mice, endometrial stromal cells proliferated rapidly and Cspg4 expression increased during decidualization. Therefore, we believe that CSPG4 plays a crucial role in the process of decidualization. The defect in decidualization caused by abnormal CSPG4 expression could lead to insufficient trophoblast invasion, ultimately contributing to the occurrence of PE.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth-factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF-mutant CSPG4-positive or -negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient-derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4-positive cells as compared to pre-therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug-resistance in melanoma cells.

Chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan originally identified as a highly immunogenic tumor antigen on the surface of melanoma cells, is associated with melanoma tumor formation and poor prognosis in certain melanomas and several other tumor types. The complex mechanisms by which CSPG4 affects melanoma progression have started to be defined, in particular the association with other cell surface proteins and receptor tyrosine kinases (RTKs) and its central role in modulating the function of these proteins. CSPG4 is essential to the growth of melanoma tumors through its modulation of integrin function and enhanced growth factor receptor-regulated pathways including sustained activation of ERK 1,2. This activation of integrin, RTK, and ERK1,2 function by CSPG4 modulates numerous aspects of tumor progression. CSPG4 expression has further been correlated to resistance of melanoma to conventional chemotherapeutics. This review outlines recent advances in our understanding of CSPG4-associated cell signaling, describing the central role it plays in melanoma tumor cell growth, motility, and survival, and explores how modifying CSPG4 function and protein-protein interactions may provide us with novel combinatorial therapies for the treatment of advanced melanoma.

Resistance to chemotherapy remains a major cause of malignant melanoma treatment failure. While recent clinical trials have shown significant patient response to chemotherapeutics targeting BRAFV600E, e.g. dabrafenib, tumors eventually become resistant and disease recurs. Development of additional novel therapies is therefore critical to better manage melanoma patients. Analysis of patient-derived melanoma lesions has revealed robust expression of the cell adhesion protein chondroitin sulfate proteoglycan 4 (CSPG4). While not prognostic due to its expression in premalignant nevi, CSPG4 has been revealed to promote melanoma progression by enhancing cell growth, migration and invasion in vitro and in vivo. Previous studies have shown CSPG4 to enhance activation of ERK1/2, PI3K and FAK, suggesting that CSPG4 likely contributes to oncogenic growth and survival through activation of a number of parallel signaling cascades. CSPG4 therefore represents a potential avenue for melanoma cell survival in response to chemotherapy. In support of this hypothesis, melanoma cells expressing endogenous or exogenous CSPG4 are more resistant to cell death induced by dabrafenib than cells lacking endogenous expression of CSPG4. Likewise, suppression of CSPG4 via siRNA reduced melanoma cell survival in response to dabrafenib. Melanoma cells expressing CSPG4 show enhanced growth in soft agar versus cells lacking CSPG4 expression, even in the presence of BRAFV600E inhibition. To determine the mechanism of CSPG4-driven resistance to BRAFV600E inhibition, activation of a number of oncogenic growth and survival pathways was interrogated. While ERK1/2 phosphorylation in the presence of BRAFV600E inhibition is unaffected by CSPG4, AKT phosphorylation is enhanced by expression of CSPG4. Moreover, this CSPG4-driven AKT activation in response to dabrafenib is dependent on PI3K, as treatment of melanoma cells with the PI3K inhibitor LY294002 eliminated increased AKT phosphorylation by CSPG4 in response to dabrafenib. CSPG4 enhances Src-FAK complex assembly, membrane lipid subdomain localization, and activation in melanoma cells through syntenin-1-dependent scaffolding. A mutant of CSPG4 engineered to eliminate lipid microdomain localization of CSPG4, termed CSPG4C2230A, was unable to recapitulate this phenotype.CSPG4C2230A also failed to protect melanoma cells from cytotoxicity or promote growth in soft agar in the presence of BRAFV600E inhibition. Altogether, these data indicate that CSPG4 confers protection against cytotoxicity induced by BRAFV600E inhibition by organizing progression-associated signalosomes involved in AKT activation, making CSPG4 an attractive target for the development of adjuvant therapies. Ongoing studies focus on determining the mechanism of CSPG4-mediated AKT activation and on developing CSPG4-targeting melanoma treatments for use in preclinical models. Citation Format: Leah E. Colvin Wanshura, Jianbo Yang, Matthew A. Price, Jennifer H. Carlson, Arkadiusz Z. Dudek, James B. McCarthy. CSPG4 mediates melanoma cell survival in response to BRAFV600E inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4058. doi:10.1158/1538-7445.AM2013-4058

BackgroundThe development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid organ and hematologic malignancies. Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target antigen that is overexpressed in multiple cancer histologies including melanoma, triple-negative breast cancer, glioblastoma, mesothelioma and sarcoma.MethodsCSPG4 expression in cancer cell lines was assayed using flow cytometry (FACS) and reverse-transcription PCR (RT-PCR). Immunohistochemistry was utilized to assay resected melanomas and normal human tissues (n = 30) for CSPG4 expression and a reverse-phase protein array comprising 94 normal tissue samples was also interrogated for CSPG4 expression. CARs were successfully constructed from multiple murine antibodies (225.28S, TP41.2, 149.53) using second generation (CD28.CD3ζ) signaling domains. CAR sequences were cloned into a gamma-retroviral vector with subsequent successful production of retroviral supernatant and PBL transduction. CAR efficacy was assayed by cytokine release and cytolysis following coculture with target cell lines. Additionally, glioblastoma stem cells were generated from resected human tumors, and CSPG4 expression was determined by RT-PCR and FACS.ResultsImmunohistochemistry demonstrated prominent CSPG4 expression in melanoma tumors, but failed to demonstrate expression in any of the 30 normal human tissues studied. Two of 94 normal tissue protein lysates were positive by protein array. CAR constructs demonstrated cytokine secretion and cytolytic function after co-culture with tumor cell lines from multiple different histologies, including melanoma, breast cancer, mesothelioma, glioblastoma and osteosarcoma. Furthermore, we report for the first time that CSPG4 is expressed on glioblastoma cancer stem cells (GSC) and demonstrate that anti-CSPG4 CAR-transduced T cells recognize and kill these GSC.ConclusionsThe functionality of multiple different CARs, with the widespread expression of CSPG4 on multiple malignancies, suggests that CSPG4 may be an attractive candidate tumor antigen for CAR-based immunotherapies using appropriate technology to limit possible off-tumor toxicity.

Background: Early development of resistance to therapy remains a major cause of cutaneous malignant melanoma treatment failure. Chondroitin sulfate proteoglycan 4 (CSPG4) is robustly expressed in a majority of metastatic melanomas. Previous work has shown CSPG4 to promote MAPK, PI3K and FAK activation, suggesting that CSPG4 represents a potential avenue for oncogenic growth and cell survival in response to chemotherapy. The PI3K-AKT-mTOR pathway regulates the cell growth and motility in the presence of nutrients. The inhibition of AKT and mTOR independently was shown to reduce the melanoma cell survival and enhanced the sensitivity of these cells to cisplatin. PF-05212384 (PKI-587) is a dual PI3K/mTOR inhibitor in clinical trials. We hereby report that the expression of CSPG4 on the melanoma cells caused them to become less responsive to the therapy with a dual PF-05212384. Material and methods: Melanoma cell lines that stably expressed CSPG4 were selected for this study. The expression of CSPG4 was knocked down by using lentivirus-transduced shRNA. Cell survival assays were performed to compare the inhibitory effects of the drug in CSPG4 expressing and non-expressing cell line. Wound healing migration assays were performed to examine the cell motility and migration. The activation of PI3K-mTOR pathway was analyzed by phosphorylation of downstream effectors for their activation using Western blotting. Results: Melanoma cells expressing active CSPG4 showed less sensitivity to the inhibitor. The average cell viability after treatment with maximum dose was 47.94% and 35.76% for CSPG4 positive 1205LU and WM1341D melanoma cell lines respectively, while the CSPG4 negative WM1552C showed an average cell viability of 19.07%. The difference in cell viability was statistically significant.(p<α=0.05)for higher doses. The suppression of CSPG4 using the shRNA sensitized the CSPG4 positive cell lines to the PI3K/mTOR inhibition. (p<α=0.05). The maximum effect was seen in the WM1341D where the cell viability was reduced at maximum dose to 26.30% as compared to 50% in non-targeting shRNA transduced control. The CSPG4 positive cell lines resisted the deactivation of p70Sk, a downstream effector of mTOR and had enhanced phosphorylation of transcription repressor 4E-BP1.At higher doses the cleavage of caspase to its activated forms, which is necessary for execution of apoptosis was reduced in the CSPG4 positive cell lines. The CSPG4 positive cells showed more motility as compared to the controls. Conclusion: This data supports the hypothesis that the CSPG4 is involved in inducing resistance in melanoma cells in response to the therapeutic challenge with the dual PI3k/mTOR inhibitor. The study highlights the need of using multiple treatment strategies to treat melanoma.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for melanoma immunotherapy, but its expression in benign melanocytic lesions and its diagnostic value remain unexplored. This study assessed CSPG4 expression in benign nevi (BN), dysplastic nevi (DN), and superficial spreading melanomas (SSM), comparing it with PRAME (PReferentially expressed Antigen in MElanoma) and evaluating the cell division cycle 7‐related protein kinase (CDC7) and the proliferation marker Ki67. Histological sections were stained using automated instruments, digitized, and analyzed using QuPath. Cohorts of BN, DN, and SSM were created, and positive cells/mm2 and H‐scores were determined. A total of 336 IHC stainings from 84 specimens were analyzed. CSPG4 expression was readily detected in SSM and was significantly stronger in DN (p = 0.005), with the highest intensity observed in BN (p < 0.001). PRAME showed the highest density of positive cells/mm2 in SSM, was significantly reduced in DN (p < 0.001), and was lowest in BN (p < 0.001). Ki67 expression was strong in SSM, moderate in DN (p = 0.62), and low in BN (p = 0.008). CDC7 expression was most intense in DN, less in SSM (p = 0.39), and weakest in BN (p = 0.002). ROC AUC values for SSM versus DN and SSM versus BN were 0.764 and 0.921 for CSPG4, 0.85 and 0.889 for PRAME, 0.735 and 0.742 for Ki67, and 0.425 and 0.767 for CDC7. While PRAME was the most reliable marker for differentiating DN and SSM, CSPG4 was superior for distinguishing BN from SSM due to its high expression in BN. However, CSPG4‐targeting therapies may trigger on‐target/off‐tumor effects due to its high expression in melanocytic nevi. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

IntroductionWe have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread.MethodsQuantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group).ResultsThe CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002).ConclusionsCell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.