Gabriella Misso

Mir-34: A New Weapon Against Cancer?

The microRNA(miRNA)-34a is a key regulator of tumor suppression. It controls the expression of a plethora of target proteins involved in cell cycle, differentiation and apoptosis, and antagonizes processes that are necessary for basic cancer cell viability as well as cancer stemness, metastasis, and chemoresistance. In this review, we focus on the molecular mechanisms of miR-34a-mediated tumor suppression, giving emphasis on the main miR-34a targets, as well as on the principal regulators involved in the modulation of this miRNA. Moreover, we shed light on the miR-34a role in modulating responsiveness to chemotherapy and on the phytonutrients-mediated regulation of miR-34a expression and activity in cancer cells. Given the broad anti-oncogenic activity of miR-34a, we also discuss the substantial benefits of a new therapeutic concept based on nanotechnology delivery of miRNA mimics. In fact, the replacement of oncosuppressor miRNAs provides an effective strategy against tumor heterogeneity and the selective RNA-based delivery systems seems to be an excellent platform for a safe and effective targeting of the tumor.

miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1.

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells.

In vivo activity of miR-34a mimics delivered by stable nucleic acid lipid particles (SNALPs) against multiple myeloma.

Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are urgently awaited. A rising body of evidence supports the notion that microRNAs (miRNAs), master regulators of eukaryotic gene expression, may exert anti-MM activity. Here, we evaluated the activity of synthetic miR-34a in MM cells. We found that transfection of miR-34a mimics in MM cells induces a significant change of gene expression with relevant effects on multiple signal transduction pathways. We detected early inactivation of pro-survival and proliferative kinases Erk-2 and Akt followed at later time points by caspase-6 and -3 activation and apoptosis induction. To improve the in vivo delivery, we encapsulated miR-34a mimics in stable nucleic acid lipid particles (SNALPs). We found that SNALPs miR-34a were highly efficient in vitro in inhibiting growth of MM cells. Then, we investigated the activity of the SNALPs miR-34a against MM xenografts in SCID mice. We observed significant tumor growth inhibition (p<0.05) which translated in mice survival benefits (p = 0.0047). Analysis of miR-34a and NOTCH1 expression in tumor retrieved from animal demonstrated efficient delivery and gene modulation induced by SNALPs miR-34a in the absence of systemic toxicity. We here therefore provide evidence that SNALPs miR-34a may represent a promising tool for miRNA-therapeutics in MM.

Cytotoxic drugs up-regulate epidermal growth factor receptor (EGFR) expression in colon cancer cells and enhance their susceptibility to EGFR-targeted antibody-dependent cell-mediated-cytotoxicity (ADCC).

Cetuximab is a human-murine chimeric IgG1 monoclonal antibody to epidermal growth factor-receptor (EGFR) which exerts synergistic antitumour interactions with several cytotoxic drugs. Therefore, it is presently recommended in combination with chemotherapy in the treatment of colon, head and neck and non-small cell lung cancer. Cetuximab has been designed to inhibit EGFR signalling; however, preclinical evidence suggests that its anti-cancer effects in vivo are also related to the ability of its human IgG1 backbone to trigger immunological mechanisms. Here we have investigated whether the exposure to different cytotoxic drugs may affect the susceptibility of colon cancer cells in vitro to cetuximab immuno-targeting and related lymphokine-activated killer (LAK)-mediated antibody-dependent cell cytotoxicity (ADCC). Five colon cancer cell lines expressing a different k-ras mutational status were evaluated for: (i) EGFR-expression, (ii) susceptibility to LAK cells and (iii) cetuximab-mediated ADCC, before and after exposure to 5-flurouracil (5-FU), gemcitabine (Gem), irinotecan (Iri) alone or in multiple two/three drug combinations. These drugs were able to up-regulate EGFR expression on the surface of all the colon cancer cell lines with a maximal effect observed few hours after the exposure to GILF regimen (Gem, Iri, Levofolinic acid and 5-FU). Chemotherapy was able to greatly enhance the sensitivity to either LAK cells or cetuximab-mediated ADCC in all the colon cancer cell lines with a mechanism independent from k-ras status. The results of our study suggest that chemotherapy may enhance cetuximab-mediated immuno-targeting and ADCC thus providing the rationale to design novel immuno-biochemotherapy regimens.

High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy

Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine—a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.

Molecular Targets for the Treatment of Multiple Myeloma

Multiple myeloma (MM) represents a suitable disease to be treated with Molecularly targeted drugs (MTDs). MM clone aberrations affect signal transduction pathways controlling both proliferation and/or cell survival. Research findings on small drugs or monoclonal antibodies (mAb) against the components of these pathways are now available and related clinical trials in MM patients are rapidly growing up. Promising results have been recently obtained with AKT inhibitors (perifosine) and mTOR inhibitors (everolimus and temsirolimus). However, the activity of these agents used alone is still limited and can be strongly increased by their combination with other drugs such as bortezomib or dexamethasone. The present review will summarize the main signaling components that can be targeted by MTDs and the most important available results derived from the clinical trials based on their use. Another important issue in the treatment of MM is the control of the related bone disease. Two main strategies can be used: i) inhibition of bone resorption and ii) promotion of bone formation. Emerging clinical data suggest that specific MTDs are able to prolong survival not only for the prevention of the skeletal-related events but also for a direct or indirect effect on the proliferation and/or survival of MM cells. A summary on the main preclinical and clinical results in this setting will be provided. In conclusion, the use of MTD in the treatment of MM is a promising approach but still far from becoming a current indication: a new dawn is arising with still unpredictable results.

Evaluation of the in vitro and in vivo antiangiogenic effects of denosumab and zoledronic acid

Denosumab (Dmab) and zoledronic acid (ZOL) are antiresorptive agents, with different mechanisms of action, that are indicated for delaying the onset of skeletal-related events in patients with bone metastases from solid tumors. Clinical and preclinical data suggest that ZOL may have also anti-angiogenic activity; however, the effects of Dmab (a fully humanized antibody against the receptor activator of nuclear factor kappa B ligand) on angiogenesis are largely unknown. The objective of this study was to compare the potential anti-angiogenic activity of Dmab with that of ZOL in preclinical models. Dmab (0.31 to 160 μM) had no effect on the viability of human MDA-MB-436 and CG5 breast cancer cells or human umbilical vein endothelial cells (HUVECs) and no effect on tubule formation or invasion of HUVECs. In contrast, ZOL (0.31 to 160 μM) decreased the viability of breast cancer and HUVECs in a time- and concentration-dependent manner and also inhibited HUVEC tubule formation and invasion. In vivo, ZOL (20 μg/mouse for three times a week for three consecutive weeks) inhibited angiogenesis in Matrigel plugs and inhibited the growth and neo-angiogenesis of CG5 xenografts in athymic nude mice. In contrast, Dmab (10 mg/Kg twice a week for 4 consecutive weeks) had no effect on Matrigel vascularization or xenograft growth in this model. These findings support the potential antiangiogenic and anticancer activity of ZOL in vitro and in vivo and further suggest that Dmab does not have antiangiogenic activity. Additional studies are needed to elucidate the potential anticancer activity of Dmab.

Transferrin-Conjugated SNALPs Encapsulating 2′-O-Methylated miR-34a for the Treatment of Multiple Myeloma

Stable nucleic acid lipid vesicles (SNALPs) encapsulating miR-34a to treat multiple myeloma (MM) were developed. Wild type or completely 2′-O-methylated (OMet) MiR-34a was used in this study. Moreover, SNALPs were conjugated with transferrin (Tf) in order to target MM cells overexpressing transferrin receptors (TfRs). The type of miR-34a chemical backbone did not significantly affect the characteristics of SNALPs in terms of mean size, polydispersity index, and zeta potential, while the encapsulation of an OMet miR-34a resulted in a significant increase of miRNA encapsulation into the SNALPs. On the other hand, the chemical conjugation of SNALPs with Tf resulted in a significant decrease of the zeta potential, while size characteristics and miR-34a encapsulation into SNALPs were not significantly affected. In an experimental model of MM, all the animals treated with SNALPs encapsulating miR-34a showed a significant inhibition of the tumor growth. However, the use of SNALPs conjugated with Tf and encapsulating OMet miR-34a resulted in the highest increase of mice survival. These results may represent the proof of concept for the use of SNALPs encapsulating miR-34a for the treatment of MM.

Phase II trial of bevacizumab and dose/dense chemotherapy with cisplatin and metronomic daily oral etoposide in advanced non-small-cell-lung cancer patients

Bevacizumab, is a humanized monoclonal antibody to vasculo-endothelial-growth-factor, with anticancer activity in non-small-cell-lung cancer (NSCLC) patients. Our previous results from a dose/finding phase I trial in NSCLC patients, demonstrated the anti-angiogenic effects and toxicity of a newest bevacizumab-based combination with fractioned cisplatin and daily oral etoposide. We designed a phase II trial to evaluate in advanced NSCLC patients the antitumor activity and the safety of this novel regimen. In particular, 45 patients (36 males and 9 females), with a mean age of 54 years, an ECOG ≤ 2, stage IIIB/IV and NSCLC (28 adenocarcinomas, 11 squamous-cell carcinomas, 2 large-cell carcinomas, 4 undifferentiated carcinomas), were enrolled. They received cisplatin (30 mg/sqm, days 1-3), oral etoposide (50 mg, days 1-15) and bevacizumab (5 mg/kg, day 3) every three weeks (mPEBev regimen). Patients who achieved an objective response or stable disease received maintenance treatment with bevacizumab in combination with erlotinib until progression. Grade I-II hematological, mucosal toxicity and alopecia were the most common adverse events. The occurrence of infections (17%), thromboembolic events (4.4%) and severe mood depression (6.7%) was also recorded. A partial response was achieved in 31 (68.8%) patients, disease remained stable in 8 (17.8%), and disease progressed in 6 (13.3%) with a progression-free-survival of 9.53 months (95%CI, 7.7-11.46). Our bio-chemotherapy regimen resulted very active in advanced NSCLC, however, the toxicity associated with the treatment requires strict selection of the patients to enroll in future studies.

Emerging pathways as individualized therapeutic target of multiple myeloma

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy, which causes significant morbidity due to organ damage and bone tissue destruction. In recent years, novel drugs have become available for MM therapy thanks to the growing knowledge of disease pathobiology. Areas covered: Intrinsic genetic lesions, as well as the bone marrow microenvironment, contribute to the activation of proliferation and survival pathways, impairment of cell death mechanisms and drug resistance. The phosphatidylinositol 3-kinase (PI3K) and the Ras/mitogen-activated protein kinase (MAPK) cascades are the signaling pathways mainly involved in the MM development. In the last decade, several molecules interfering with growth and survival promoting signaling have been developed. Expert opinion: Despite the availability of novel therapeutics, MM still evolves into a drug-resistant phase and most patients die of progressive disease. Therefore, there is an urgent need of novel therapeutic strategies. Among a plethora of new investigational agents, microRNA (miRNA) represents the basis for the design of novel therapeutic strategies which basically rely on miRNA inhibition or miRNA replacement approaches and take benefit respectively from the use of miRNA inhibitors or synthetic miRNAs as well as from lipid-based nanoparticles as carriers for in vivo delivery.