BACKGROUND:The most prevalent solid tumors in young men are testicular germ cell tumors (TGCTs), and embryonal carcinoma is the most common subtype among non-seminomatous germ cell tumors (NSGCTs). Despite the excellent cure rates of cisplatin-based chemotherapy, resistance develops in 15%-30% of patients with metastatic cancer, which results in a poor prognosis. The overexpression of the High Mobility Group A2 (HMGA2) protein has been linked to treatment resistance and cancer aggressiveness. It is well known that this protein promotes carcinogenesis. OBJECTIVE:The purpose of this work was to investigate the functional role of HMGA2 in EC cell migration, survival, and proliferation, focusing on its role as a potential therapeutic target in cisplatin-resistant ECs. MATERIALS AND METHODS:We employed human EC cell lines EP2102 and GCT27, as well as the cisplatin-resistant (CisR) versions of these cell lines that were produced by prolonged drug treatment. siRNA transfection was used to suppress HMGA2 expression. Growth curve and colony formation tests were used to measure cell proliferation. Apoptosis was assessed by Annexin V staining followed by flow cytometry, cell cycle distribution was analyzed by flow cytometry, and cell migration was detected by Boyden Chamber Assays. RESULTS:In both parental and resistant EC cell lines, HMGA2 knockdown markedly decreased proliferation. After HMGA2 knockdown, flow cytometric analysis revealed S phase arrest. Apoptosis was significantly elevated, especially in cells that were resistant to cisplatin. In addition, all HMGA2-depleted cell lines showed decreased migration. These impacts were true for both the GCT27 and EP2102 models. DISCUSSION:The data suggest that HMGA2 is necessary to preserve the EC cells' malignant characteristics. Its silencing interferes with several cancer-related functions, including motility, survival, and cell cycle progression. These results indicate HMGA2's participation in chemoresistance mechanisms and are consistent with its role in other solid cancers. CONCLUSION:Our results indicate a role of HMGA2 in the EC, because its inhibition reduces cell malignant characteristics, and may represent a viable therapeutic target to improve the prognosis of CisR TGCTs.

INTRODUCTION:The most prevalent solid cancer in young adult males is testicular germ cell tumors (TGCTs), which are becoming more and more common globally. Approximately 20% of patients with metastatic disease relapse or develop resistance to cisplatin-based chemotherapy, despite its high effectiveness, underscoring the need for alternative therapies. AREAS COVERED:With an emphasis on new molecular targets and biomarkers, this review describes developments in TGCT pathophysiology and treatment. Tyrosine kinase receptors, transcription factors, elements of the DNA damage response, and cell cycle regulators are important oncogenic drivers. CDK inhibitors, PARP inhibitors, and Aurora kinase antagonists are promising early-stage agents. Meanwhile, noninvasive liquid biopsy techniques are being made possible by biomarkers like CLDN6, cfDNA/ctDNA, and the miR-371 ~ 373 cluster, which may enhance disease monitoring, risk assessment, and diagnosis. EXPERT OPINION:Although the majority of TGCT cases have positive results, cisplatin-refractory disease continues to be a significant therapeutic challenge. A route to precision medicine is provided by molecular profiling and biomarker-driven approaches. However, implementation requires clinical validation, patient selection, and standardization. The landscape of TGCT treatment may change over the next 10 years as a result of the integration of targeted therapies and molecular diagnostics, which may greatly increase survival rates while lowering long-term toxicity.

Background/Objectives: The UbcH10 protein plays an important role in a variety of human malignancies, including thyroid, breast, ovarian, and colorectal carcinomas. It has been previously reported that UbcH10 is overexpressed in non-small cell lung cancer (NSCLC) compared to normal lungs and that its expression is directly and inversely correlated with the mutational status of p53 and EGFR, respectively. Methods: We transfected lung cancer cells with wild-type and mutant forms of EGFR, modulated the expression of UbcH10 and p53, and treated these cells with tyrosine kinase inhibitor (TKI) erlotinib. Using Western blotting, we evaluated the expression of UbcH10 induced by EGFR and p53. Finally, we employed immunohistochemistry to assess the levels of UbcH10 expression in a subset of NSCLC patients receiving TKI therapy. Results: We reported a possible modulation of UbcH10 expression by the overexpression of wild-type and mutant EGFR in H460 lung cancer cells, potentially through p53. The enforced expression of UbcH10 in cells transfected with mutant EGFR suggested a potential increase in resistance to erlotinib treatment. Finally, immunohistochemical analysis of samples from NSCLC patients with mutant EGFR indicated a possible connection between UbcH10 expression levels and progression-free survival. Conclusions: In NSCLC, UbcH10 may play a role in the regulation of TKI response via a molecular pathway potentially involving p53 and EGFR. However, further research is needed to fully understand this mechanism.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal neoplasms in humans, and just limited progresses have been made to extend patient survival and decrease ATC-associated mortality. Thus, the identification of novel therapeutic strategies for treating ATC is needed. Recently, our group has identified two proteins with oncogenic activity, namely HMGA1 and EZH2, with pivotal roles in ATC cancer progression. Therefore, we tested the ability of trabectedin, a HMGA1-targeting drug, and GSK126, an inhibitor of EZH2 enzymatic activity, to impair cell viability of four ATC-derived cell lines. In the present study, we first confirmed the overexpression of HMGA1 and EZH2 in all ATC-derived cell lines and tissues compared to the normal primary thyroid cells and tissues. Then, treatment of the ATC cell lines with trabectedin and GSK126 resulted in a drastic induction of apoptotic cell death, which increased when the ATC cell lines were treated with a combination of both drugs. Conversely, normal primary human thyroid cells did not show any significant reduction in their viability when exposed to the same drugs. Noteworthy, both drugs induced the deregulation of EZH2- and HMGA1-controlled genes. Altogether, these findings propose the combination of trabectedin and GSK126 as possible novel strategy for ATC therapy.

Supplementary Figure Legends 1-2 from Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Supplementary figures S1-S7. Fig S1. Klhl14-AS is down-regulated in thyroid cancer in a MAPK-dependent manner; Fig S2. A. Rat Klhl14-AS alternative transcripts as reported by Ensembl (Rnor_6). B, C Klhl14-AS was knocked-down in FRTL-5 using LNA Gapmers targeting the region indicated in A by the dashed box (Khl14-AS LNA) or control LNA (ctr LNA). Klhl14-AS and Ki67 expression were measured by qRT-pcr. D Human KLHL14-AS transcript as reported by Ensembl (GRCh38.p12). E KLHL14-AS was knocked-down in Nthy-ori 3-1 using LNA Gapmers targeting the region indicated in D by the dashed box (KHL14-AS LNA) or control LNA (ctr LNA). KLHL14-AS expression was measured through qRT-pcr. All data are shown as means {plus minus} SD. *** p < 0.001; Fig S3: miR-182-5p and miR-20a-5p Responsive Elements in the highly conserved region of Klhl14-AS; Fig S4: Klhl14-AS does not bind let7a; Fig S5. Pax8 expression is repressed in thyroid transformaRon models; Fig S6. Pax8 or Bcl2 overexpression have no effects on thyroid differenRaRon gene expression; Fig S7. GEPIA-annotated thyroid cancer data.

Supplementary Table 4 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Thyroid cancer is the most prevalent endocrine malignancy and comprises a wide range of lesions subdivided into differentiated (DTC) and undifferentiated thyroid cancer (UTC), mainly represented by the anaplastic thyroid carcinoma (ATC). This is one of the most lethal malignancies in humankind leading invariably to patient death in few months. Then, a better comprehension of the mechanisms underlying the development of ATC is required to set up new therapeutic approaches. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides in length that do not code for proteins. They show a strong regulatory function at both transcriptional and post-transcriptional level and are emerging as key players in regulating developmental processes. Their aberrant expression has been linked to several biological processes, including cancer, making them potential diagnostic and prognostic markers. We have recently analyzed the lncRNA expression profile in ATC through a microarray technique and have identified rhabdomyosarcoma 2-associated transcript ( RMST ) as one of the most downregulated lncRNA in ATC. RMST has been reported to be deregulated in a series of human cancers, to play an anti-oncogenic role in triple-negative breast cancer, and to modulate neurogenesis by interacting with SOX2. Therefore, these findings prompted us to investigate the role of RMST in ATC development. In this study we show that RMST levels are strongly decreased in ATC, but only slightly in DTC, indicating that the loss of this lncRNA could be related to the loss of the differentiation and high aggressiveness. We also report a concomitant increase of SOX2 levels in the same subset of ATC, that inversely correlated with RMST levels, further supporting the RMST/SOX2 relationship. Finally, functional studies demonstrate that the restoration of RMST in ATC cells reduces cell growth, migration and the stemness properties of ATC stem cells. In conclusion, these findings support a critical role of RMST downregulation in ATC development.

Supplementary Table 3 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Legends of the Supplementary Figures

Supplementary Figure S1 from Haploinsufficiency of the Hmga1 Gene Causes Cardiac Hypertrophy and Myelo-Lymphoproliferative Disorders in Mice

Supplementary Table 5 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Supplementary Figure 1 from Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Supplementary tables S1-S5. Tab S1: Oligonucleo1des used as primers for qRT-PCR; Tab S2: Oligonucleo1des used for cloning in either reporter or expression vectors. Only gene-specific sequences are reported; Tab S3: Klhl-14-AS specific oligonucleo1des used in RNA pull-down experiments; Tab S4: PITA analysis results of miRNA binding sites. The conserved region of Klhl14-AS was analyzed by using PITA algorithm for the presence of binding sites for miRNAs. Only miRNAs predicted to bind on the conserved sequences in all the species are shown; Tab S5: Rat Pax8 and Bcl-2 MREs. PutaZve MREs on the Bcl2 and Pax8 transcripts were idenZfied by searching for rno-miR-182 and rno-miR-20a-5p canonical (7-8- nt) and marginal (6-nt) seed-matched sites using a custom Perl script.

Supplementary Table 2 from A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Bladder cancer (BC) is the tenth most common cancer, with urothelial carcinoma representing about 90% of all BC, including neoplasms and carcinomas of different grades of malignancy. Urinary cytology has a significant role in BC screening and surveillance, although it has a low detection rate and high dependence on the pathologist's experience. The currently available biomarkers are not implemented into routine clinical practice due to high costs or low sensitivity. In recent years, the role of lncRNAs in BC has emerged, even though it is still poorly explored. We have previously shown that the lncRNAs Metallophosphoesterase Domain-Containing 2 Antisense RNA 1 (MPPED2-AS1), Rhabdomyosarcoma-2 Associated Transcript (RMST), Kelch-like protein 14 antisense (Klhl14AS) and Prader Willi/Angelman region RNA 5 (PAR5) are involved in the progression of different types of cancers. Here, we investigated the expression of these molecules in BC, first by interrogating the GEPIA database and observing a different distribution of expression levels between normal and cancer specimens. We then measured them in a cohort of neoplastic bladder lesions, either benign or malignant, from patients with suspicion of BC undergoing transurethral resection of bladder tumor (TURBT). The total RNA from biopsies was analyzed using qRT-PCR for the expression of the four lncRNA genes, showing differential expression of the investigated lncRNAs between normal tissue, benign lesions and cancers. In conclusion, the data reported here highlight the involvement of novel lncRNAs in BC development, whose altered expression could potentially affect the regulatory circuits in which these molecules are involved. Our study paves the way for testing lncRNA genes as markers for BC diagnosis and/or follow-up.

Supplementary Figure 2 from Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

FOSL2 expression in papillary thyroid carcinoma