Discoidin Domain Receptor Tyrosine Kinase 1 (DDR1) Is a Novel Therapeutic Target in Liposarcoma: A Tissue Microarray Study.

BACKGROUND: Liposarcoma is the most commonly diagnosed subtype of soft tissue sarcoma. As these tumors often arise near vital organs and neurovascular structures, complete resection can be challenging; consequently, recurrence rates are high. Additionally, available chemotherapeutic agents have shown limited benefit and substantial toxicities. There is, therefore, a clear and unmet need for novel therapeutics for liposarcoma. Discoidin domain receptor tyrosine kinase 1 (DDR1) is involved in adhesion, proliferation, differentiation, migration, and metastasis in several cancers. However, the expression and clinical importance of DDR1 in liposarcoma are unknown. QUESTIONS/PURPOSES: The purposes of this study were to assess (1) the expression, (2) the association between DDR1 and survival, and (3) the functional roles of DDR1 in liposarcoma. METHODS: The correlation between DDR1 expression in tumor tissues and clinicopathological features and survival was assessed via immunohistochemical staining of a liposarcoma tissue microarray. It contained 53 samples from 42 patients with liposarcoma and 11 patients with lipoma. The association between DDR1 and survival in liposarcoma was analyzed by Kaplan-Meier plots and log-rank tests. The DDR1 knockout liposarcoma cell lines were generated by CRISPR-Cas9 technology. The DDR1-specific and highly selective DDR1 inhibitor 7RH was applied to determine the impact of DDR1 expression on liposarcoma cell growth and proliferation. In addition, the effect of DDR1 inhibition on liposarcoma growth was further accessed in a three-dimensional cell culture model to mimic DDR1 effects in vivo. RESULTS: The results demonstrate elevated expression of DDR1 in all liposarcoma subtypes relative to benign lipomas. Specifically, high DDR1 expression was seen in 55% (23 of 42) of liposarcomas and no benign lipomas. However, DDR1 expression was not found to be associated with poor survival in patients with liposarcoma. DDR1 knockout or treatment of 7RH showed decreased liposarcoma cell growth and proliferation. CONCLUSION: DDR1 is aberrantly expressed in liposarcoma, and it contributes to several markers of oncogenesis in these tumors. CLINICAL RELEVANCE: This work supports DDR1 as a promising therapeutic target in liposarcoma.

Establishment and Characterization of a Novel Dedifferentiated Chondrosarcoma Cell Line DDCS2.

BACKGROUND: The dedifferentiated variant of chondrosarcoma is highly aggressive and carries an especially grim prognosis. While chemotherapeutics has failed to benefit patients with dedifferentiated chondrosarcoma significantly, preclinical chemosensitivity studies have been limited by a scarcity of available cell lines. There is, therefore, an urgent need to expand the pool of available cell lines. METHODS: We report the establishment of a novel dedifferentiated chondrosarcoma cell line DDCS2, which we isolated from the primary tumor specimen of a 60-year-old male patient. We characterized its short tandem repeat (STR) DNA profile, growth potential, antigenic markers, chemosensitivity, and oncogenic spheroid and colony-forming capacity. RESULTS: DDCS2 showed a spindle to polygonal shape and an approximate 60-hour doubling time. STR DNA profiling revealed a unique genomic identity not matching any existing cancer cell lines within the ATCC, JCRB, or DSMZ databases. There was no detectable contamination with another cell type. Western blot and immunofluorescence assays were consistent with a mesenchymal origin, and our MTT assay revealed relative resistance to conventional chemotherapeutics, which is typical of a dedifferentiated chondrosarcoma. Under ex vivo three-dimensional (3D) culture conditions, the DDCS2 cells produced spheroid patterns similar to the well-established CS-1 and SW1353 chondrosarcoma cell lines. CONCLUSION: Our findings confirm DDCS2 is a novel model for dedifferentiated chondrosarcoma and therefore adds to the limited pool of current cell lines urgently needed to investigate the chemoresistance within this deadly cancer.

Cyclin-dependent kinase 9 (CDK9) is a novel prognostic marker and therapeutic target in ovarian cancer.

Despite surgical and chemotherapeutic advances over the past few decades, the prognosis for ovarian cancer remains very poor. Although cyclin-dependent kinase (CDK) 9 has an established pathogenic role in various cancers, its function in ovarian cancer remains poorly defined. The purpose of this study was to evaluate the expression of CDK9 and its therapeutic potential in ovarian cancer. CDK9 expression was determined by immunohistochemistry in a unique ovarian cancer tissue microarray constructed with paired primary, metastatic, and recurrent tumor tissues from 26 ovarian cancer patients. CDK9 was highly expressed in human ovarian cancer cell lines and was also elevated in metastatic and recurrent ovarian tumor tissue compared with patient-matched primary ovarian tumor tissue. In addition, increased CDK9 significantly correlated with poor patient prognosis. Inhibition of CDK9 by small interfering RNA or CDK9 inhibitor functionally suppressed RNA transcription elongation, induced apoptosis, and reduced proliferation of ovarian cancer cells. Inhibition of CDK9 also suppressed ovarian cancer cell spheroid growth, clonogenicity formation, and migration activity. Our results reveal CDK9 as a novel prognostic biomarker and a promising therapeutic target for preventing metastasis and recurrence while also improving the overall clinical outcome for ovarian cancer patients.-Wang, J., Dean, D. C., Hornicek, F. J., Shi, H., Duan, Z. Cyclin-dependent kinase 9 (CDK9) is a novel prognostic marker and therapeutic target in ovarian cancer.

Cyclin-dependent kinase 12 (CDK12) in chordoma: prognostic and therapeutic value.

PURPOSE: To determine the cyclin-dependent kinase 12 (CDK12) expression in chordoma patient tissues and cell lines, its correlation with oncologic outcomes, and its function in chordoma cell proliferation. METHODS: A chordoma tissue microarray was constructed from fifty-six patient specimens and examined by immunohistochemistry to measure CDK12 expression and its correlation to patient clinical characteristics and survival. CDK12 expression in chordoma cell lines and patient tissues was evaluated via western blot. CDK12 specific small interfering RNA (siRNA) was applied to determine whether its inhibition attenuated chordoma cell growth and proliferation. RESULTS: CDK12 was expressed in the majority of chordoma specimens, with notably higher expression in patients with recurrent or metastatic disease. High CDK12 expression was an independent prognostic predictor for shorter overall and progression-free survival in chordoma by univariate and multivariate analysis. Western blot analysis revealed that CDK12 was also highly expressed in chordoma cell lines, with CDK12 specific small interfering RNA (siRNA) mediated knockdown decreasing proliferation and inducing apoptosis. Mechanistically, inhibition of CDK12 decreased phosphorylation of RNA polymerase II (RNAP II) and the anti-apoptotic proteins Survivin and Mcl-1. CONCLUSION: High expression of CDK12 is an independent predictor of poor prognosis in chordoma. Inhibition of CDK12 significantly decreased chordoma cell proliferation and induced apoptosis. Our results support CDK12 as a novel prognostic biomarker and therapeutic target in chordoma.

Exosomes promote pre-metastatic niche formation in ovarian cancer.

Ovarian cancer is one of the most common gynecological malignancies. Upon initial diagnosis, the majority of patients present with widespread metastatic growth within the peritoneal cavity. This metastatic growth occurs in stages, with the formation of a pre-metastatic niche occurring prior to macroscopic tumor cell invasion. Exosomes released by the primary ovarian tumor are small extracellular vesicles which prepare the distant tumor microenvironment for accelerated metastatic invasion. They regulate intercellular communication between tumor cells and normal stroma, cancer-associated fibroblasts, and local immune cells within the tumor microenvironment. In this review, we highlight the emerging roles of ovarian cancer exosomes as coordinators of pre-metastatic niche formation, biomarkers amenable to liquid biopsy, and targets of chemotherapy.

From genomics to metabolomics: emerging metastatic biomarkers in osteosarcoma.

Although the investigation into biomarkers specific for pulmonary metastasis within osteosarcoma (OS) has recently expanded, their usage within the clinic remains sparse. The current screening protocol after any OS diagnosis includes a chest CT scan; however, metastatic lung nodules frequently go undetected and remain the primary cause of death in OS. Recently, screening technologies such as liquid biopsy and next-generation sequencing have revealed a promising array of biomarkers with predictive and diagnostic value for the pulmonary metastasis associated with OS. These biomarkers draw from genomics, transcriptomics, epigenetics, and metabolomics. When assessed in concert, their utility is most promising as OS is a highly heterogeneous cancer. Accordingly, there has been an expansion of clinical trials not only aimed at further demonstrating the significance of these individual biomarkers but to also reveal which therapies resolve the pulmonary metastasis once detected. This review will focus on the recently discovered and novel metastatic biomarkers within OS, their molecular and cellular mechanisms, the expansion of humanized OS mouse models amenable to their testing, and the associated clinical trials aimed at managing the metastatic phase of OS.

RNA sequencing (RNA-Seq) and its application in ovarian cancer.

Despite the surgical and chemotherapeutic advances over the past few decades, ovarian cancer remains the leading cause of gynecological cancer-related mortality. The absence of biomarkers in early detection and the development of drug resistance are principal causes of treatment failure in ovarian cancer. Recent progress in RNA sequencing (RNA-Seq) with Next Generation Sequencing technology has expanded the understanding of the molecular pathogenesis of ovarian cancer. As compared to previous hybridization-based microarray and Sanger sequence-based methods, RNA-Seq provides multiple layers of resolutions and transcriptome complexity, with less background noise and a broader dynamic range of RNA expression. Beyond quantifying gene expression, the data generated by RNA-Seq accelerates the identification of alternatively spliced genes, fusion genes, mutations/SNPs, allele-specific expression, novel transcripts and non-coding RNAs. RNA-Seq has been successfully applied in ovarian cancer research for earlier detection, ascertaining pathological origin, and defining the aberrant genes and dysregulated molecular pathways across patient groups. This review outlines the distinct advantages of RNA-Seq compared to other transcriptomics methods and its recent applications in ovarian cancer.

Role of cyclin-dependent kinases (CDKs) in hepatocellular carcinoma: Therapeutic potential of targeting the CDK signaling pathway.

Liver cancer is the fourth leading cause of cancer related mortality in the world, with hepatocellular carcinoma (HCC) representing the most common primary subtype. Two-thirds of HCC patients have advanced disease when diagnosed, and for these patients, treatment strategies remain limited. In addition, there is a high incidence of tumor recurrence after surgical resection with the current treatment regimens. The development of novel and more effective agents is required. Cyclin-dependent kinases (CDKs) constitute a family of 21 different protein kinases involved in regulating cell proliferation, apoptosis, and drug resistance, and are evaluated in preclinical and clinical trials as chemotherapeutics. To summarize and discuss the therapeutic potential of targeting CDKs in HCC, recent published articles identified from PubMed were comprehensively reviewed. The key words included hepatocellular carcinoma, cyclin-dependent kinases, and CDK inhibitors. This review focuses on the emerging evidence from studies describing the genetic and functional aspects of CDKs in HCC. We also present an overview of CDK inhibitors that have shown efficacy in laboratory studies of HCC. Although many of the studies assessing CDK-targeting therapies in HCC are at the preclinical stage, there is significant evidence that CDK inhibitors used alone or in combination with established chemotherapy drugs could have significant applications in HCC.

Erratum: ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage.

[This corrects the article on p. 1577 in vol. 12, PMID: 35530299.].

Expression and Clinical Significance of High-Mobility Group AT-hook 2 (HMGA2) in Osteosarcoma.

OBJECTIVE: Although high-mobility group AT-hook 2 (HMGA2) has been shown to have crucial roles in the pathogenesis and metastasis of various malignancies, its expression and significance in osteosarcoma remain unknown. Here we evaluate the expression, clinical prognostic value, and overall function of HMGA2 in osteosarcoma. METHODS: Sixty-nine osteosarcoma patient specimens within a tissue microarray (TMA) were analyzed by immunohistochemistry for HMGA2 expression. Demographics and clinicopathological information including age, gender, tumor location, metastasis, recurrence, chemotherapy response, follow-up time, and disease status were also collected. After validation of expression, we determined whether there was a correlation between HMGA2 expression and patient clinicopathology. HMGA2 expression was also evaluated in osteosarcoma cell lines and patient tissues by Western blot, we analyzed the expression of HMGA2 in the human osteosarcoma cell lines MG63, 143B, U2OS, Saos-2, MNNG/HOS, and KHOS. HMGA2-specific siRNA and clonogenic assays were then used to determine the effect of HMGA2 inhibition on osteosarcoma cell proliferation, growth, and chemosensitivity. RESULTS: HMGA2 expression was elevated in the osteosarcoma patient specimens and human osteosarcoma cell lines. HMGA2 was differentially expressed in human osteosarcoma cell lines. Specifically, a relatively high expression of HMGA2 was present in KHOS, MNNG/HOS, 143B and a relatively low expression was in MG63, U2OS as well as Saos-2. HMGA2 expression is correlated with metastasis and shorter overall survival. High HMGA2 expression is an independent predictor of poor osteosarcoma prognosis. There was no significant correlation between HMGA2 expression and the age, gender, or tumor site of the patient. HMGA2 expression is predominantly within the nucleus. The expression of HMGA2 also directly correlated to neoadjuvant chemoresistance. There was a significant reduction of HMGA2 expression in the siRNA transfection group. After the use of siRNA, the proliferation of osteosarcoma cells is decreased and the chemosensitivity of osteosarcoma cells is significantly increased. CONCLUSION: Our study supports HMGA2 as a potential prognostic biomarker and therapeutic target in osteosarcoma.

SMARCB1 expression is a novel diagnostic and prognostic biomarker for osteosarcoma.

BACKGROUND: Although weak SWI/SNF related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1) expression is a known diagnostic and prognostic biomarker in several malignancies, its expression and clinical significance in osteosarcoma remain unknown. The aim of the present study was to investigate SMARCB1 expression in osteosarcoma and its clinical significance with respect to chemosensitivity and prognosis. METHODS: We obtained 114 specimens from 70 osteosarcoma patients to construct a tissue microarray (TMA) and assess SMARCB1 protein expression via immunohistochemistry (IHC). The mRNA expression of SMARCB1 was in-silico analyzed using open-access RNA sequencing (RNA-Seq) and clinicopathological data provided by the Therapeutically Applicable Research to Generate Effective Treatments on Osteosarcoma (TARGET-OS) project. The correlations between SMARCB1 expression and clinical features were statistically analyzed. RESULTS: Weak SMARCB1 expression occurred in 70% of the osteosarcoma patient specimens in the TMA, and significantly correlated with poor neoadjuvant response as well as shorter overall and progression-free survival (PFS). In addition, mRNA in-silico analysis confirmed that SMARCB1 expression correlates with chemotherapeutic response and prognosis in osteosarcoma patients. CONCLUSION: To our knowledge, the present study is the first to analyze SMARCB1 expression in osteosarcoma. SMARCB1 may serve as a novel diagnostic and prognostic biomarker in osteosarcoma.

ATR inhibition sensitizes liposarcoma to doxorubicin by increasing DNA damage.

Liposarcomas account for approximately 20% of all adult sarcomas and have limited therapeutic options outside of surgery. Inhibition of ataxia-telangiectasia and Rad3 related protein kinase (ATR) has emerged as a promising chemotherapeutic strategy in various cancers. However, its activation, expression, and function in liposarcoma remain unkown. In this study, we investigated the expression, function, and potential of ATR as a therapeutic target in liposarcoma. Activation and expression of ATR in liposarcoma was analyzed by immunohistochemistry, which was further explored for correlation with patient clinical characteristics. ATR-specific siRNA and the ATR inhibitor VE-822 were applied to determine the effect of ATR inhibition on liposarcoma cell proliferation and anti-apoptotic activity. Migration activity and clonogenicity were examined using wound healing and clonogenic assays. ATR (p-ATR) was overexpressed in 88.1% of the liposarcoma specimens and correlated with shorter overall survival in patients. Knockdown of ATR via specific siRNA or inhibition with VE-822 suppressed liposarcoma cell growth, proliferation, migration, colony-forming ability, and spheroid growth. Importantly, ATR inhibition significantly and synergistically enhanced liposarcoma cell line chemosensitivity to doxorubicin. Our findings support ATR as critical to liposarcoma proliferation and doxorubicin resistance. Therefore, the addition of ATR inhibition to a standard doxorubicin regimen is a potential treatment strategy for liposarcoma.

Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

Synovial sarcoma is typical aggressive malignant without satisfactory treatment outcome in adult series. Cyclin-dependent kinases (CDKs) in transcription have been considered promising molecular targets in cancer. Among these, CDK7 has been shown to play important roles in the pathogenesis of malignancies. However, the modulation mechanism of CDK7-regulated transcription in synovial sarcoma is unknown. In the present study, we aim to determine the expression and function of CDK7 in the transcription cycle of RNA polymerase II (RNAP II), and evaluate its prognostic and therapeutic significance in synovial sarcoma. Results showed that overexpression of CDK7 correlates with higher clinical stage and grade, and worse outcomes in clinic. High CDK7 expression was confirmed in all tested human synovial sarcoma cell lines and CDK7 was largely localized to the cell nucleus. Downregulation through siRNA or inhibition with the CDK7-targeting agent BS-181 exhibited dose-dependent cytotoxicity and prevented cell colony formation. Western blots demonstrated that inhibition of CDK7 paused transcription by a reduction of RNAP II phosphorylation. Blocking CDK7-dependent transcriptional addiction was accompanied by promotion of apoptosis. Furthermore, the CDK7-specific inhibitor reduced 3D spheroid formation and migration of synovial sarcoma. Collectively, our findings highlight the role of CDK7-dependent transcriptional addiction in human synovial sarcoma. CDK7-specific cytotoxic agents are therefore promising novel treatment options for synovial sarcoma.

Cyclin-dependent kinase 7 (CDK7) is an emerging prognostic biomarker and therapeutic target in osteosarcoma.

BACKGROUND: Overexpression of cyclin-dependent kinase 7 (CDK7) is a well-known pathogenic feature of various malignancies and a sign of a more dismal prognosis. As relatively little is known about CDK7 in osteosarcoma, we elected to evaluate its expression, prognostic value, and function. METHODS: We began by analyzing the publicly available data sets on CDK7 expression, including RNA sequencing data from the Therapeutically Applicable Research to Generate Effective Treatments on Osteosarcoma (TARGET-OS) and the Gene Expression database of Normal and Tumor tissues 2 (GENT2). The correlation between patient tissue CDK7 expression and their clinicopathological features and prognosis was assessed via immunohistochemical staining of a unique tissue microarray constructed from osteosarcoma specimens. Furthermore, we analyzed CDK7 expression in osteosarcoma cell lines and tissues by Western blot. CDK7-specific siRNA and a highly-selective CDK7 inhibitor, BS-181, were applied to determine the function of CDK7 on osteosarcoma cell growth and proliferation. In addition, the effect of CDK7 inhibition on clonogenicity was evaluated using a clonogenic assay, and a 3D cell culture model was used to mimic CDK7 effects in an in vivo environment. RESULTS: Our results demonstrate that higher CDK7 expression significantly correlates with recurrence, metastasis, and shorter overall survival in osteosarcoma patients. Therapeutically, we show that CDK7 knockdown with siRNA or selective inhibition with BS-181 decreases proliferation and induces apoptosis of osteosarcoma cells. CONCLUSION: This study supports CDK7 overexpression as an independent predictor of poor prognosis and promising therapeutic target for osteosarcoma.

T-LAK cell-originated protein kinase (TOPK): an emerging prognostic biomarker and therapeutic target in osteosarcoma.

T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK) is an emerging target with critical roles in various cancers; however, its expression and function in osteosarcoma remain unexplored. We evaluated TOPK expression using RNA sequencing and gene expression data from public databases (TARGET-OS, CCLE, GTEx, and GENT2) and immunohistochemistry in an osteosarcoma tissue microarray (TMA). TOPK gene expression was significantly higher in osteosarcoma than normal tissues and directly correlated with shorter overall survival. TOPK was overexpressed in 83.3% of the osteosarcoma specimens within our TMA and all osteosarcoma cell lines, whereas normal osteoblast cells had no aberrant expression. High expression of TOPK associated with metastasis, disease status, and shorter overall survival. Silencing of TOPK with small interfering RNA (siRNA) decreased cell viability, and inhibition with the selective inhibitor OTS514 suppressed osteosarcoma cell proliferation, migration, colony-forming ability, and spheroid growth. Enhanced chemotherapeutic sensitivity and a synergistic effect were also observed with the combination of OTS514 and either doxorubicin or cisplatin in osteosarcoma cell lines. Taken together, our study demonstrated that TOPK is a potential prognostic biomarker and therapeutic target for osteosarcoma treatment.

Fibroblast elongation and dendritic extensions in constrained versus unconstrained microtissues.

Cytoskeletal tension is fundamental to many biological processes, including germ layer sorting during embryogenesis [Krieg et al., 2008]. In vitro, such tension influences cell sorting in self-assembled, 3D microtissues and can be of sufficient magnitude to cause complex-shaped microtissue failure [Dean et al., 2007]. To examine the process of failure under cell-derived tension, we subjected normal human fibroblasts (NHFs) to directed self-assembly [Dean et al., 2007] in micro-molds designed to yield self-constraining microtissues. As cells contracted in this assay, the constrained microtissues narrowed, thinned and ultimately failed at their midpoints. By adding small numbers of GFP+ cells, changes in cell movement and morphology were assessed and compared to those of unconstrained microtissues. We found that cells formed numerous dendritic extensions within an hour of self-assembly and retracted these extensions as they elongated up to 30 times their initial diameter ( approximately 600 microm) just prior to failure. Surprisingly, significant coordination in cell motility was observed over large distances within microtissues. Pharmacologic interventions showed that failure was myosin II and Rho kinase dependent and inhibition of failure resulted in shorter cells with greater numbers of extensions. These findings further our understanding of cellular self-assembly and introduce the use of GFP+ cells with directed self-assembly as a scaffold-free analogue to fibroblast-populated collagen gels (FPCGs).

Prognostic Significance of Cyclin E1 Expression in Patients With Chordoma: A Clinicopathological and Immunohistochemical Study.

PURPOSE: Chordomas are rare, slow-growing sarcomas without any accepted prognostic biomarkers. Owing to their proximity to critical neurovascular structures, discovering predictive biomarkers in chordoma has been a significant research effort because it may potentially reduce risky therapies in patients with less aggressive tumors. In response, because cyclin E1 overexpression correlates with patient prognosis in several malignancies, we investigated its expression in chordoma and whether it informs patient prognosis. METHODS: Seventy-five chordoma patient specimens were enrolled in a tissue microarray (TMA) to evaluate cyclin E1 expression via immunohistochemical staining. Western blot was used to assess cyclin E1 expression in chordoma cell lines and fresh tissues. We then correlated cyclin E1 staining intensity in the TMA to clinicopathological features and chordoma patient outcomes. RESULTS: Sixty-three percent of the chordoma patient specimens in the TMA, fifty-six percent of the fresh chordoma tissues, and all chordoma cell lines showed high cyclin E1 expression. In TMA analysis, cyclin E1 expression positively correlated to chordoma patient disease status. By survival analysis, high cyclin E1 expression was an independent prognostic risk factor for chordoma patients along with advanced disease status and positive surgical margin. CONCLUSION: Cyclin E1 is a promising biomarker predicting chordoma patient prognosis.

Cyclin E1 is a prognostic biomarker and potential therapeutic target in osteosarcoma.

While amplified expressed cyclin E1 is a well-known tumorigenic factor and prognostic biomarker in several malignancies, its prognostic predictive potential and function in osteosarcoma is poorly understood. Here we reveal discrete expression pattern, correlation to clinicopathological characteristics and prognosis and overall function of cyclin E1 in osteosarcoma. Sixty-nine osteosarcoma patient tumor specimens were enrolled to construct a tissue microarray to evaluate cyclin E1 expression through immunohistochemical staining. Cyclin E1 expression in osteosarcoma cell lines and fresh tissues was assessed by Western blot. Cyclin E1 gene expression was evaluated using RNA sequencing data acquired from the public database. We correlated staining intensity to clinical characteristics. Cyclin E1 small interfering RNA was used to determine the effect of cyclin E1 silencing on osteosarcoma cell proliferation and chemotherapeutic sensitivity. Sixty-one percent of the osteosarcoma patient specimens in the tissue microarray had high cyclin E1 expression. Cyclin E1 gene was significantly highly expressed in osteosarcoma tissues and cell lines compared to normal tissues. The expression of cyclin E1 positively correlated with disease status, and inversely correlated to prognosis and response to neoadjuvant chemotherapy. The expression of cyclin E1 was an independent prognostic factor for osteosarcoma patients. In addition, silencing cyclin E1 expression in osteosarcoma cells significantly inhibited cell proliferation and increased sensitivity to chemotherapeutics. We conclude that cyclin E1 is overexpressed in osteosarcoma and is a promising biomarker for prognosis and chemotherapeutic response. We confirm aberrant cyclin E1 expression is a potent therapeutic target in osteosarcoma, and its selective inhibition is a rational treatment strategy for osteosarcoma.

Aberrant CDK9 expression within chordoma tissues and the therapeutic potential of a selective CDK9 inhibitor LDC000067.

Objectives: Chordomas are slow-growing malignancies that commonly affect vital neurological structures. These neoplasms are highly resistant to current chemotherapeutic regimens and often recur after surgical intervention. Therefore, there is an urgent need to identify molecular targets and more robust drugs to improve chordoma patient outcomes. It is well accepted that cyclin-dependent protein kinase 9 (CDK9) has tumorigenic roles in various cancers; however, the expression and significance of CDK9 in chordoma remains unknown. Methods: Expression of CDK9 in chordoma cell lines and tumor tissues was examined by Western blot and immunohistochemistry (IHC). The correlation between CDK9 expression in patient tissues and clinical prognosis was analyzed. The functional roles of CDK9 in chordoma were investigated after the addition of small interfering RNA (siRNA) and CDK9 inhibitor (LDC000067). Cell growth and proliferation were assessed with MTT and clonogenic assays. The effect of CDK9 inhibition on chordoma cells was further evaluated with a three-dimensional (3D) cell culture model which mimics the in vivo environment. Results: CDK9 was expressed in both chordoma cell lines and chordoma tissues. High- expression of CDK9 correlated with recurrence and poor outcomes for chordoma patients. CDK9 silencing with siRNA decreased growth and proliferation of chordoma cells and lowered levels of Mcl-1 and RNA polymerase II (RNAP II) phosphorylation. Pharmacological inhibition of CDK9 with the small molecular inhibitor LDC000067 reduced cell growth, supported apoptosis, suppressed cell colony formation in a clonogenic assay, and decreased spheroid growth in 3D culture. Conclusion: We demonstrate that CDK9 expression in chordoma correlates with patient outcome, and, when inhibited, chordoma cell growth and proliferation significantly decreases. Taken together, these results support CDK9 as an emerging potential target in chordoma therapy.

Chimeric antigen receptor T (CAR-T) cell immunotherapy for sarcomas: From mechanisms to potential clinical applications.

Survival rates for sarcoma patients have plateaued in the past few decades and remain especially grim for those with recurrent or metastatic disease. This has prompted investigation into novel immunotherapies for sarcomas, especially after their recent and well-recognized successes in other cancers. One such modality, the Chimeric Antigen Receptor (CAR) T Cell therapy, has shown promising results in treating B-cell lymphoma and acute lymphoblastic leukemia. This novel therapy functions by fusing a specific antibody derived single-chain variable fragment (scFv) with a T-cell which recognizes a specific tumor-associated antigen (TAA). Several sarcoma-associated antigens (SAA) amenable to CAR-T cell treatment have recently emerged with encouraging results. These include human epidermal growth factor receptor 2 (HER2), disialoganglioside (GD2), interleukin 11 Receptor Subunit Alpha (IL-11RA), fibroblast activation protein (FAP), B7-H3, CD44v6, insulin-like growth factor 1 receptor (IGF-1R), and tyrosine kinase orphan-like receptor 1 (ROR1). Given the limitations of current medical therapies, novel treatment strategies are urgently needed. As a sarcoma treatment modality, CAR-T cell therapy is highly promising and continues to draw interest especially as new clinical trials emerge. Here we review recent breakthrough CAR-T cell studies in sarcoma, the targets which define them, and approaches to minimizing host cytotoxicity.