Iris Maria Forte

MicroRNA-29b-1 impairs in vitro cell proliferation, self‑renewal and chemoresistance of human osteosarcoma 3AB-OS cancer stem cells

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR-29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we showed a potent downregulation of miR-29b. In this study, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated the role of miR-29b-1 in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS cells, in vitro. We found that, miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis, it did not influence their migratory and invasive capacities, thus suggesting a context-depending role of miR-29b-1. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b-1 as a novel therapeutic agent might offer benefits for OS treatment.

New pyrazolo[3,4-d]pyrimidine SRC inhibitors induce apoptosis in mesothelioma cell lines through p27 nuclear stabilization

Malignant mesothelioma (MM) is a highly aggressive tumor of the serous membranes for which there is currently no effective curative modality. Recent data suggest that hyperactivation of the tyrosine kinase SRC has a key role in MM development and therefore this kinase represents an important molecular target for MM therapy. We tested new pyrazolo[3,4-d]pyrimidine SRC inhibitors on a panel of MM cell lines expressing the active form of SRC. These SRC inhibitors exerted a significant proapoptotic effect on MM cells without affecting the normal mesothelial cell line MET-5A, supporting a possible use of these SRC inhibitors for a safe treatment of MM. We also showed that SRC inhibitor-induced apoptosis occurred concomitantly with an increase in the nuclear stability of the cyclin-dependent kinase inhibitor p27. This finding is remarkable considering that loss of nuclear p27 expression is a well-established adverse prognostic factor in MM, and p27 nuclear localization is crucial for its tumor-suppressive function. Consistently, SRC inhibition seems to promote the increase in p27 nuclear level also by inactivating the AKT kinase and downregulating cyclin D1, which would otherwise delay p27 nuclear import and provoke its cytoplasmic accumulation. To determine whether p27 stabilization has a direct role in apoptosis induced by SRC inhibition, we stably silenced the CDKN1B gene, encoding p27, in MSTO-211H and REN mesothelioma cells by transduction with lentiviral vectors expressing short hairpin RNAs against the CDKN1B transcript. Strikingly, p27 silencing was able to suppress the apoptosis induced by these SRC inhibitors in both MM cell lines, suggesting that p27 has a crucial proapoptotic role in MM cells treated with SRC inhibitors. Our findings reveal a new mechanism, dependent on p27 nuclear stabilization, by which SRC inhibition can induce apoptosis in MM cells and provide a new rationale for the use of SRC inhibitors in MM therapy.

Modeling human osteosarcoma in mice through 3AB‐OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012.

Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma

Malignant mesothelioma, a very aggressive tumor associated to asbestos exposure, is expected to increase in incidence, and unfortunately, no curative modality exists. Reactivation of p53 is a new attractive antitumoral strategy. p53 is rarely mutated in mesothelioma, but it is inactivated in most tumors by the lack of p14ARF. Here, we evaluated the feasibility of this approach in pleural mesothelioma by testing RITA and nutlin-3, two molecules able to restore p53 function through a different mechanism, on a panel of mesothelioma cell lines representing the epithelioid (NCI-H28, NCI-H2452, IST-MES 2), biphasic (MSTO-211H), and sarcomatoid (NCI-H2052) histotypes compared with the normal mesothelial HMC-hTERT. RITA triggered robust caspase-dependent apoptosis specifically in epithelioid and biphasic mesothelioma cell lines, both through wild-type and mutant p53, concomitant to p21 downregulation. Conversely, nutlin-3 induced a p21-dependent growth arrest, rather than apoptosis, and was slightly toxic on HMC-hTERT. Interestingly, we identified a previously undetected point mutation of p53 (p.Arg249Ser) in IST-MES 2, and showed that RITA is also able to reactivate this p53 mutant protein and its apoptotic function. RITA reduced tumor growth in a MSTO-211H-derived xenograft model of mesothelioma and synergized with cisplatin, which is the mainstay of treatment for this tumor. Our data indicate that reactivation of p53 and concomitant p21 downregulation effectively induce cell death in mesothelioma, a tumor characterized by a high intrinsic resistance to apoptosis. Altogether, our findings provide the preclinical framework supporting the use of p53-reactivating agents alone, or in combination regimens, to improve the outcome of patients with mesothelioma.

Antitumor activity of new pyrazolo[3,4-d]pyrimidine SRC kinase inhibitors in Burkitt lymphoma cell lines and its enhancement by WEE1 inhibition

Burkitt lymphoma (BL) is a highly aggressive B cell neoplasm. Although intensive polychemotherapy regimens have proven very effective, they are associated with significant toxicities. Therefore, more rational therapies that selectively target the molecular abnormalities of BL are needed. Recent data suggest that the tyrosine kinase SRC could represent a therapeutic target for BL. We found that new pyrazolo[3,4-d]pyrimidine SRC inhibitors exerted a significant cytotoxic effect and induced apoptosis on two BL cell lines, as determined by MTS assays, cytofluorimetric analyses and caspase 3 assay. Notably, our SRC inhibitors proved to be more effective than the well-known SRC inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine] in BL cells. Moreover, our small molecules induced a G2/M arrest in BL cells through a possible new mechanism, whereby SRC inhibition hinders an AKT-WEE1-cyclin-dependent kinase 1 (CDK1) axis, leading to inhibition of CDK1, the main trigger of entry into mitosis. By using a small-molecule inhibitor of WEE1, a crucial CDK1 negative regulator, we were able to shift the balance toward apoptosis rather than growth arrest and enhance the efficacy of the SRC inhibitors, suggesting a possible use of these selective drugs in combination for a safe and efficient treatment of BL.

Abrogating G2/M checkpoint through WEE1 inhibition in combination with chemotherapy as a promising therapeutic approach for mesothelioma

Malignant mesothelioma (MM) is a very aggressive asbestos-related neoplasm of the serous membranes, whose incidence is increasing worldwide. Although the introduction of new drug combinations, such as cisplatin plus pemetrexed/gemcitabine, has determined an improvement in the patient quality of life, MM remains a universally fatal disease. The observation that key G1/S checkpoint regulators are often functionally inactivated in MM prompted us to test whether the use of G2/M checkpoint inhibitors, able to sensitize G1/S checkpoint-defective cancer cells to DNA-damaging agents, could be successful in MM. We treated six MM cell lines, representative of different histotypes (epithelioid, biphasic, and sarcomatoid), with cisplatin in combination with MK-1775, an inhibitor of the G2/M checkpoint kinase WEE1. We observed that MK-1775 enhanced the cisplatin cytotoxic effect in all MM cell lines, except the sarcomatoid cell line, which is representative of the most aggressive histotype. As expected, the enhancement in cisplatin toxicity was accompanied by a decrease in the inactive phosphorylated form of cyclin-dependent kinase 1 (CDK1), a key substrate of WEE1, which is indicative of G2/M checkpoint inactivation. Consistently, we also observed a decrease in G2/M accumulation and an increase in mitotic entry of DNA-damaged cells and apoptosis, probably due to the loss of the cell ability to arrest cell cycle in response to DNA damage, irrespectively of p53 mutational status. Notably, this treatment did not increase cisplatin cytotoxicity on normal cells, thus suggesting a possible use of MK-1775 in combination with cisplatin for a safe and efficient treatment of epithelioid and biphasic MM.

SRC Family Kinase Inhibition Through a New Pyrazolo[3,4‐d]Pyrimidine Derivative as a Feasible Approach for Glioblastoma Treatment

Glioblastoma (GB) is the most common and aggressive primary tumor of the central nervous system. The current standard of care for GB consists of surgical resection, followed by radiotherapy combined with temozolomide chemotherapy. However, despite this intensive treatment, the prognosis remains extremely poor. Therefore, more effective therapies are urgently required. Recent studies indicate that SRC family kinases (SFKs) could represent promising molecular targets for GB therapy. Here, we challenged four GB cell lines with a new selective pyrazolo[3,4‐d]pyrimidine derivative SFK inhibitor, called SI221. This compound exerted a significant cytotoxic effect on GB cells, without significantly affecting non‐tumor cells (primary human skin fibroblasts), as evaluated by MTS assay. We also observed that SI221 was more effective than the well‐known SFK inhibitor PP2 in GB cells. Notably, despite the high intrinsic resistance to apoptosis of GB cells, SI221 was able to induce this cell death process in all the GB cell lines, as observed through cytofluorimetric analysis and caspase‐3 assay. SI221 also exerted a long‐term inhibition of GB cell growth and was able to reduce GB cell migration, as shown by clonogenic assay and scratch test, respectively. Moreover, through in vitro pharmacokinetic assays, SI221 proved to have a high metabolic stability and a good potential to cross the blood brain barrier, which is an essential requirement for a drug intended to treat brain tumors. Therefore, despite the need of developing strategies to improve SI221 solubility, our results suggest a potential application of this selective SFK inhibitor in GB therapy. J. Cell. Biochem. 116: 856–863, 2015.

SRC Family Kinase Inhibition in Ewing Sarcoma Cells Induces p38 MAP Kinase-Mediated Cytotoxicity and Reduces Cell Migration

Ewing sarcoma (ES) is a highly aggressive bone and soft tissue cancer, representing the second most common primary malignant bone tumor in children and adolescents. Although the development of a multimodal therapy, including both local control (surgery and/or radiation) and systemic multidrug chemotherapy, has determined a significant improvement in survival, patients with metastatic and recurrent disease still face a poor prognosis. Moreover, considering that ES primarily affects young patients, there are concerns about long‐term adverse effects of the therapy. Therefore, more rational strategies, targeting specific molecular alterations underlying ES, are required. Recent studies suggest that SRC family kinases (SFKs), which are aberrantly activated in most cancer types, could represent key therapeutic targets also for ES. Here, we challenged ES cell lines with a recently developed selective SFK inhibitor (a pyrazolo[3,4‐d]pyrimidine derivative, called SI221), which was previously shown to be a valuable proapoptotic agent in other tumor types while not affecting normal cells. We observed that SI221 significantly reduced ES cell viability and proved to be more effective than the well‐known SFK inhibitor PP2. SI221 was able to induce apoptosis in ES cells and also reduced ES cell clonogenic potential. Furthermore, SI221 was also able to reduce ES cell migration. At the molecular level, our data suggest that SFK inhibition through SI221 could reduce ES cell viability at least in part by hindering an SFK‐NOTCH1 receptor‐p38 mitogen‐activated protein kinase (MAPK) axis. Overall, our study suggests a potential application of specific SFK inhibition in ES therapy. J. Cell. Physiol. 232: 129–135, 2017.

Let‐7d miRNA Shows Both Antioncogenic and Oncogenic Functions in Osteosarcoma‐Derived 3AB‐OS Cancer Stem Cells

Osteosarcoma (OS), an aggressive highly invasive and metastatic bone‐malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self‐renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let‐7d microRNA was downregulated in the 3AB‐OS‐CSCs, derived from the human OS‐MG63 cells. Here, we aimed to assess whether let‐7d modulation affected tumorigenic and stemness properties of these OS‐CSCs. We found that let‐7d‐overexpression reduced cell proliferation by decreasing CCND2 and E2F2 cell‐cycle‐activators and increasing p21 and p27 CDK‐inhibitors. Let‐7d also decreased sarcosphere‐and‐colony forming ability, two features associated with self‐renewing, and it reduced the expression of stemness genes, including Oct3/4, Sox2, Nanog, Lin28B, and HMGA2. Moreover, let‐7d induced mesenchymal‐to‐epithelial‐transition, as shown by both N‐Cadherin‐E‐cadherin‐switch and decrease in vimentin. Surprisingly, such switch was accompanied by enhanced migratory/invasive capacities, with a strong increase in MMP9, CXCR4 and VersicanV1. Let‐7d‐ overexpression also reduced cell sensitivity to apoptosis induced by both serum‐starvation and various chemotherapy drugs, concomitant with decrease in caspase‐3 and increase in BCL2 expression. Our data suggest that let‐7d in 3AB‐OS‐CSCs could induce plastic‐transitions from CSCs‐to‐non‐CSCs and vice‐versa. To our knowledge this is the first study to comprehensively examine the expression and functions of let‐7d in OS‐CSCs. By showing that let‐7d has both tumor suppressor and oncogenic functions in this context, our findings suggest that, before prospecting new therapeutic strategies based on let‐7d modulation, it is urgent to better define its multiple functions. J. Cell. Physiol. 231: 1832–1841, 2016.

pRb2/p130 localizes to the cytoplasm in diffuse gastric cancer

pRb2/p130 is a key tumor suppressor, whose oncosuppressive activity has mainly been attributed to its ability to negatively regulate cell cycle by interacting with the E2F4 and E2F5 transcription factors. Indeed, pRb2/p130 has been found altered in various cancer types in which it functions as a valuable prognostic marker. Here, we analyzed pRb2/p130 expression in gastric cancer tissue samples of diffuse histotype, in comparison with their normal counterparts. We found a cytoplasmic localization of pRb2/p130 in cancer tissue samples, whereas, in normal counterparts, we observed the expected nuclear localization. pRb2/p130 cytoplasmic delocalization can lead to cell cycle deregulation, but considering the emerging involvement of pRb2/p130 in other key cellular processes, it could contribute to gastric tumorigenesis also through other mechanisms. Our data support the necessity of further investigations to verify the possibility of using pRb2/p130 as a biomarker or potential therapeutic target for diffuse gastric cancer. J. Cell. Physiol. 230: 802–805, 2015.