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The promising expectations about personalized medicine have opened the path to routine large-scale sequencing and increased the importance of genetic counseling for hereditary cancers, among which hereditary breast and ovary cancers (HBOC) have a major impact. High-throughput sequencing, or Next-Generation Sequencing (NGS), has improved cancer patient management, ameliorating diagnosis and treatment decisions. In addition to its undeniable clinical utility, NGS is also unveiling a large number of variants that we are still not able to clearly define and classify, the variants of uncertain significance (VUS), which account for about 40% of total variants. At present, VUS use in the clinical context is challenging. Medical reports may omit this kind of data and, even when included, they limit the clinical utility of genetic information. This has prompted the scientific community to seek easily applicable tests to accurately classify VUS and increase the amount of usable information from NGS data. In this review, we will focus on NGS and classification systems for VUS investigation, with particular attention on HBOC-related genes and in vitro functional tests developed for ameliorating and accelerating variant classification in cancer.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease with a high death rate mainly due to the metastatic spread. The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival (OS) in EOC. However, the basis of this association remains elusive. We show that in vivo MDM4 reduces intraperitoneal dissemination of EOC cells, independently of p53 and an immune-competent background. By 2D and 3D assays, MDM4 impairs the early steps of the metastatic process. A 3D-bioprinting system, ad hoc developed by co-culturing EOC spheroids and endothelial cells, showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Consistent with these data, high MDM4 levels protect mice from ovarian cancer-related death and, importantly, correlate with increased 15 y OS probability in large data set analysis of 1656 patients. Proteomic analysis of EOC 3D-spheroids revealed decreased protein synthesis and mTOR signaling, upon MDM4 expression. Accordingly, MDM4 does not further inhibit cell migration when its activity towards mTOR is blocked by genetic or pharmacological approaches. Importantly, high levels of MDM4 reduced the efficacy of mTOR inhibitors in constraining cell migration. Overall, these data demonstrate that MDM4 impairs EOC metastatic process by inhibiting mTOR activity and suggest the usefulness of MDM4 assessment for the tailored application of mTOR-targeted therapy.

Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. HIPK2 phosphorylates oncosuppressor p53 for apoptotic activation. In addition, also p53-independent apoptotic pathways are regulated by HIPK2 and can be exploited for anticancer purpose too. Therefore, HIPK2 activity is considered a central switch in targeting tumor cells toward apoptosis upon genotoxic damage and the preservation and/or restoration of HIPK2 function is crucial for an efficient tumor response to therapies. As a proof of principle, HIPK2 knockdown impairs p53 function, induces chemoresistance, angiogenesis, and tumor growth in vivo , on the contrary, HIPK2 overexpression activates apoptotic pathways, counteracts hypoxia, inhibits angiogenesis, and induces chemosensitivity both in p53-dependent and -independent ways. The role of HIPK2 in restraining tumor development was also confirmed by studies with HIPK2 knockout mice. Recent findings demonstrated that HIPK2 inhibitions do exist in tumors and depend by several mechanisms including HIPK2 cytoplasmic localization, protein degradation, and loss of heterozygosity (LOH), recapitulating the biological outcome obtained by RNA interference studies in tumor cells, such as p53 inactivation, resistance to therapies, apoptosis inhibition, and tumor progression. These findings may lead to new diagnostic and therapeutic approaches for treating cancer patients. This review will focus on the last updates about HIPK2 contribution in tumorigenesis and cancer treatment.

Background SMC1A is a subunit of the cohesin complex that participates in many DNA- and chromosome-related biological processes. Previous studies have established that SMC1A is involved in cancer development and in particular, is overexpressed in chromosomally unstable human colorectal cancer (CRC). This study aimed to investigate whether SMC1A could serve as a therapeutic target for CRC. Methods At first, we studied the effects of either SMC1A overexpression or knockdown in vitro. Next, the outcome of SMC1A knocking down (alone or in combination with bevacizumab, a monoclonal antibody against vascular endothelial growth factor) was analyzed in vivo. Results We found that SMC1A knockdown affects cell proliferation and reduces the ability to grow in anchorage-independent manner. Next, we demonstrated that the silencing of SMC1A and the combo treatment were effective in increasing overall survival in a xenograft mouse model. Functional analyses indicated that both treatments lead to atypical mitotic figures and gene expression dysregulation. Differentially expressed genes were implicated in several pathways including gene transcription regulation, cellular proliferation, and other transformation-associated processes. Conclusions These results indicate that SMC1A silencing, in combination with bevacizumab, can represent a promising therapeutic strategy for human CRC.

Background Pancreatic ductal adenocarcinoma (PDAC) features KRAS mutations in approximately 90% of human cases and excessive stromal response, termed desmoplastic reaction. Oncogenic KRAS drives pancreatic carcinogenesis by acting on both epithelial cells and tumor microenvironment (TME). We have previously shown that Homeodomain-Interacting Protein Kinase 2 (HIPK2) cooperates with KRAS in sustaining ERK1/2 phosphorylation in human colorectal cancers. Here, we investigated whether HIPK2 contributes to oncogenic KRAS-driven tumorigenesis in vivo, in the onset of pancreatic cancer. Methods We employed an extensively characterized model of KRAS G12D -dependent preinvasive PDAC, the Pdx1-Cre;LSL-KRas G12D/ + (KC) mice. In these mice, HIPK2 was inhibited by genetic knockout in the pancreatic epithelial cells (KCH −/− ) or by pharmacologic inactivation with the small molecule 5-IodoTubercidin (5-ITu). The development of preneoplastic acinar-to-ductal metaplasia (ADM), intraepithelial neoplasia (PanIN), and their associated desmoplastic reaction were analyzed. Results In Hipk2 -KO mice (KCH −/− ), ERK phosphorylation was lowered, the appearance of ADM was slowed down, and both the number and pathologic grade of PanIN were reduced compared to Hipk2 -WT KC mice. The pancreatic lesion phenotype in KCH −/− mice was characterized by abundant collagen fibers and reduced number of αSMA + and pSTAT3 + desmoplastic cells. These features were reminiscent of the recently described human “deserted” sub-TME, poor in cells, rich in matrix, and associated with tumor differentiation. In contrast, the desmoplastic reaction of KC mice resembled the “reactive” sub-TME, rich in stromal cells and associated with tumor progression. These observations were confirmed by the pharmacologic inhibition of HIPK2 in KC mice. Conclusion This study demonstrates that HIPK2 inhibition weakens oncogenic KRAS activity and pancreatic tumorigenesis providing a rationale for testing HIPK2 inhibitors to mitigate the incidence of PDAC development in high-risk individuals. Graphical Abstract

Background Cancer cells are characterized by chromosomal instability (CIN) and it is thought that errors in pathways involved in faithful chromosome segregation play a pivotal role in the genesis of CIN. Cohesin forms a large protein ring that binds DNA strands by encircling them. In addition to this central role in chromosome segregation, cohesin is also needed for DNA repair, gene transcription regulation and chromatin architecture. Though mutations in both cohesin and cohesin-regulator genes have been identified in many human cancers, the contribution of cohesin to cancer development is still under debate. Methods Normal mucosa, early adenoma, and carcinoma samples deriving from 16 subjects affected by colorectal cancer (CRC) were analyzed by OncoScan for scoring both chromosome gains and losses (CNVs) and loss of heterozygosity (LOH). Then the expression of SMC1A was analyzed by immunochemistry in 66 subjects affected by CRC. The effects of SMC1A overexpression and mutated SMC1A were analyzed in vivo using immunocompromised mouse models. Finally, we measured global gene expression profiles in induced-tumors by RNA-seq. Results Here we showed that SMC1A cohesin core gene was present as extra-copies, mutated, and overexpressed in human colorectal carcinomas. We then demonstrated that cohesin overexpression led to the development of aggressive cancers in immunocompromised mice through gene expression dysregulation. Conclusion Collectively, these results support a role of defective cohesin in the development of human colorectal cancer.

Background Despite the promise of dual BRAF/MEK inhibition as a therapy for BRAF-mutant (BRAF-mut) melanoma, heterogeneous responses have been observed in patients, thus predictors of benefit from therapy are needed. We have previously identified semaphorin 6A (SEMA6A) as a BRAF-mut-associated protein involved in actin cytoskeleton remodeling. The purpose of the present study is to dissect the role of SEMA6A in the biology of BRAF-mut melanoma, and to explore its predictive potential towards dual BRAF/MEK inhibition. Methods SEMA6A expression was assessed by immunohistochemistry in melanoma cohort RECI1 ( N  = 112) and its prognostic potential was investigated in BRAF-mut melanoma patients from DFCI and TCGA datasets ( N  = 258). The molecular mechanisms regulated by SEMA6A to sustain tumor aggressiveness and targeted therapy resistance were investigated in vitro by using BRAF-mut and BRAF-wt melanoma cell lines, an inducible SEMA6A silencing cell model and a microenvironment-mimicking fibroblasts-coculturing model. Finally, SEMA6A prediction of benefit from dual BRAF/MEK inhibition was investigated in melanoma cohort RECI2 ( N  = 14). Results Our results indicate higher protein expression of SEMA6A in BRAF-mut compared with BRAF-wt melanoma patients and show that SEMA6A is a prognostic indicator in BRAF-mut melanoma from TCGA and DFCI patients cohorts. In BRAF-mut melanoma cells, SEMA6A coordinates actin cytoskeleton remodeling by the RhoA-dependent activation of YAP and dual BRAF/MEK inhibition by dabrafenib+trametinib induces SEMA6A/RhoA/YAP axis. In microenvironment-mimicking co-culture condition, fibroblasts confer to melanoma cells a proliferative stimulus and protect them from targeted therapies, whereas SEMA6A depletion rescues the efficacy of dual BRAF/MEK inhibition. Finally, in BRAF-mut melanoma patients treated with dabrafenib+trametinib, high SEMA6A predicts shorter recurrence-free interval. Conclusions Overall, our results indicate that SEMA6A contributes to microenvironment-coordinated evasion of melanoma cells from dual BRAF/MEK inhibition and it might be a good candidate predictor of short-term benefit from dual BRAF/MEK inhibition.

Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody. Molecular characterization showed that Aurora-B directly binds and phosphorylates H2B at Ser32 while indirectly recruits HIPK2 through the central spindle components MgcRacGAP and PRC1. Thus, among different cytokinetic functions, Aurora-B separately recruits HIPK2 and H2B to the midbody and these activities contribute to faithful cytokinesis.

Background Liquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release. Methods We measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection ( N  = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release. Results Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding. Conclusions Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

Background Lamins, key nuclear lamina components, have been proposed as candidate risk biomarkers in different types of cancer but their accuracy is still debated. AKTIP is a telomeric protein with the property of being enriched at the nuclear lamina. AKTIP has similarity with the tumor susceptibility gene TSG101. AKTIP deficiency generates genome instability and, in p53 −/− mice, the reduction of the mouse counterpart of AKTIP induces the exacerbation of lymphomas. Here, we asked whether the distribution of AKTIP is altered in cancer cells and whether this is associated with alterations of lamins. Methods We performed super-resolution imaging, quantification of lamin expression and nuclear morphology on HeLa, MCF7, and A549 tumor cells, and on non-transformed fibroblasts from healthy donor and HGPS (LMNA c.1824C > T p.Gly608Gly) and EDMD2 (LMNA c.775 T > G) patients. As proof of principle model combining a defined lamin alteration with a tumor cell setting, we produced HeLa cells exogenously expressing the HGPS lamin mutant progerin that alters nuclear morphology. Results In HeLa cells, AKTIP locates at less than 0.5 µm from the nuclear rim and co-localizes with lamin A/C. As compared to HeLa, there is a reduced co-localization of AKTIP with lamin A/C in both MCF7 and A549. Additionally, MCF7 display lower amounts of AKTIP at the rim. The analyses in non-transformed fibroblasts show that AKTIP mislocalizes in HGPS cells but not in EDMD2. The integrated analysis of lamin expression, nuclear morphology, and AKTIP topology shows that positioning of AKTIP is influenced not only by lamin expression, but also by nuclear morphology. This conclusion is validated by progerin-expressing HeLa cells in which nuclei are morphologically altered and AKTIP is mislocalized. Conclusions Our data show that the combined alteration of lamin and nuclear morphology influences the localization of the tumor-associated factor AKTIP. The results also point to the fact that lamin alterations per se are not predictive of AKTIP mislocalization, in both non-transformed and tumor cells. In more general terms, this study supports the thesis that a combined analytical approach should be preferred to predict lamin-associated changes in tumor cells. This paves the way of next translational evaluation to validate the use of this combined analytical approach as risk biomarker.