Publications by authors named "Carla Azzurra Amoreo"

24 Publications

  • Page 1 of 1

HIPK2 is a potential predictive marker of a favorable response for adjuvant chemotherapy in stage II colorectal cancer.

Oncol Rep 2021 Mar 24;45(3):899-910. Epub 2020 Dec 24.

Unit of Cellular Networks and Molecular Therapeutic Targets, IRCCS Regina Elena National Cancer Institute, I‑00144 Rome, Italy.

Colorectal cancer (CRC) is the third most frequently diagnosed type of cancer worldwide. Stage II CRC accounts for ~25% all CRC cases and their management after surgical resection remains a clinical dilemma due to the lack of reliable criteria for identifying patients who may benefit from adjuvant chemotherapy. Homeodomain‑interacting protein kinase 2 (HIPK2), a multifunctional kinase involved in numerous signaling pathways, serves several key roles in cell response to different types of stresses, including chemotherapy‑induced genotoxic damage. In the present study, immunohistochemistry was performed for HIPK2 on a tissue microarray of primary human tumor samples from 84 patients with stage II CRC, treated (30 patients) or not treated (54 patients) with adjuvant chemotherapy, and sequenced for the TP53 gene, a key HIPK2 target in genotoxic damage response. It was observed that, regardless of the TP53 gene status, a high percentage of HIPK2+ cells was associated with therapeutic vulnerability in stage II CRC, suggesting a contribution of HIPK2 to drug‑response in vivo. For the in vitro characterization, HIPK2 was depleted in human CRC cells by CRISPR/Cas9 or RNA interference. HIPK2‑proficient and HIPK2‑defective cells were evaluated for their response to 5‑fluorouracil (5‑FU) and oxaliplatin (OXA). The results revealed that HIPK2 depletion induced resistance to 5‑FU and OXA, and that this resistance was not overcome by brusatol, an inhibitor of the antioxidant response regulator nuclear factor erythroid 2‑related factor 2 (NRF2), which is frequently overexpressed in CRC. By contrast, cell sensitivity to 5‑FU and OXA was further induced by brusatol supplementation in HIPK2‑proficient cells, further supporting the contribution of HIPK2 in chemotherapy response. Overall, the present results suggested that HIPK2 may be a potential predictive marker for adjuvant‑treated stage II CRC and for prospective therapy with NRF2 modulators.
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http://dx.doi.org/10.3892/or.2020.7912DOI Listing
March 2021

Assessment of HER2 Protein Overexpression and Gene Amplification in Renal Collecting Duct Carcinoma: Therapeutic Implication.

Cancers (Basel) 2020 Nov 12;12(11). Epub 2020 Nov 12.

Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, via Orabona 4, 70126 Bari, Italy.

Collecting duct carcinoma (CDC) is rare and aggressive histology of kidney cancers. Although different therapeutic approaches have been tested, the 2-year survival remains very poor. Since CDC exhibits overlapping features with urothelial carcinoma, the analysis of shared molecular alterations could provide new insights into the understanding of this rare disease and also therapeutic options. We collected 26 CDC cases, and we assessed HER2 protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in-situ hybridization (FISH) according to 2018 ASCO/CAP HER2-testing recommendations. Six out of twenty-six (23%) tumors showed HER2 positive staining. In particular, 3+ score was present in 2/6 cases (33%), 2+ in 3/6 cases (50%) and 1+ in 1/6 cases (17%). The 6 HER2+ tumors were also analyzed by FISH to assess gene copy number. One out of six CDC with IHC 3+ was also HER2 amplified, showing an average HER2 copy number ≥4.0 (10.85) and a HER2/CEP17 ratio ≥ (5.63), while the 5/6 cases were HER2 negative. Based on the 2018 ASCO/CAP guidelines overall, 2/26 CDC cases (8%) were HER2+. The present study provides evidence for testing, in future studies, HER2 to assess its clinical value as a novel target for the treatment of this highly malignant cancer.
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http://dx.doi.org/10.3390/cancers12113345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697829PMC
November 2020

BRAF status modulates Interelukin-8 expression through a CHOP-dependent mechanism in colorectal cancer.

Commun Biol 2020 Oct 1;3(1):546. Epub 2020 Oct 1.

Medical Oncology 1, IRCCS - Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Inflammation might substantially contribute to the limited therapeutic success of current systemic therapies in colorectal cancer (CRC). Amongst cytokines involved in CRC biology, the proinflammatory chemokine IL-8 has recently emerged as a potential prognostic/predictive biomarker. Here, we show that BRAF mutations and PTEN-loss are associated with high IL-8 levels in CRC models in vitro and that BRAF/MEK/ERK, but not PI3K/mTOR, targeting controls its production in different genetic contexts. In particular, we identified a BRAF/ERK2/CHOP axis affecting IL-8 transcription, through regulation of CHOP subcellular localization, and response to targeted inhibitors. Moreover, RNA Pol II and an open chromatin status in the CHOP-binding region of the IL-8 gene promoter cooperate towards increased IL-8 expression, after a selective BRAF inhibition. Overall, our data show that IL-8 production is finely and differentially regulated depending on the tumor genetic context and might be targeted for therapeutic purposes in molecularly defined subgroups of CRC patients.
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http://dx.doi.org/10.1038/s42003-020-01263-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530707PMC
October 2020

TRF2 and VEGF-A: an unknown relationship with prognostic impact on survival of colorectal cancer patients.

J Exp Clin Cancer Res 2020 Jun 15;39(1):111. Epub 2020 Jun 15.

Oncogenomic and Epigenetic Unit, IRCCS - Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Background: Colorectal cancer is one of most common tumors in developed countries and, despite improvements in treatment and diagnosis, mortality rate of patients remains high, evidencing the urgent need of novel biomarkers to properly identify colorectal cancer high-risk patients that would benefit of specific treatments. Recent works have demonstrated that the telomeric protein TRF2 is over-expressed in colorectal cancer and it promotes tumor formation and progression through extra-telomeric functions. Moreover, we and other groups evidenced, both in vitro on established cell lines and in vivo on tumor bearing mice, that TRF2 regulates the vascularization mediated by VEGF-A. In the present paper, our data evidence a tight correlation between TRF2 and VEGF-A with prognostic relevance in colorectal cancer patients.

Methods: For this study we sampled 185 colorectal cancer patients surgically treated and diagnosed at the Regina Elena National Cancer Institute of Rome and investigated the association between the survival outcome and the levels of VEGF-A and TRF2.

Results: Tissue microarray immunohistochemical analyses revealed that TRF2 positively correlates with VEGF-A expression in our cohort of patients. Moreover, analysis of patients' survival, confirmed in a larger dataset of patients from TCGA, demonstrated that co-expression of TRF2 and VEGF-A correlate with a poor clinical outcome in stage I-III colorectal cancer patients, regardless the mutational state of driver oncogenes.

Conclusions: Our results permitted to identify the positive correlation between high levels of TRF2 and VEGF-A as a novel prognostic biomarker for identifying the subset of high-risk colorectal cancer patients that could benefit of specific therapeutic regimens.
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http://dx.doi.org/10.1186/s13046-020-01612-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294609PMC
June 2020

Cross-sectional analysis of circulating tumor DNA in primary colorectal cancer at surgery and during post-surgery follow-up by liquid biopsy.

J Exp Clin Cancer Res 2020 Apr 20;39(1):69. Epub 2020 Apr 20.

Oncogenomics Division, Eurofins Genoma Group, Via Castel Giubileo, 11, 00138, Rome, Italy.

Background: Liquid biopsy (LB) in early-stage, non-metastatic colorectal cancer (CRC) must be sensitive enough to detect extremely low circulating tumor DNA (ctDNA) levels. This challenge has been seldom and non-systematically investigated.

Methods: Next generation sequencing (NGS) and digital PCR (dPCR) were combined to test tumor DNAs (tDNAs) and paired ctDNAs collected at surgery from 39 patients, 12 of whom were also monitored during the immediate post-surgery follow up. Patients treated for metastatic disease (n = 14) were included as controls.

Results: NGS and dPCR concordantly (100% agreement) called at least one single nucleotide variant (SNV) in 34 tDNAs, estimated differences in allelic frequencies being negligible (±1.4%). However, despite dPCR testing, SNVs were only detectable in 15/34 (44.1%) ctDNAs from patients at surgery, as opposed to 14/14 (100%) metastatic patients. This was likely due to striking differences (average 10 times, up to 500) in ctDNA levels between groups. NGS revealed blood-only SNVs, suggesting spatial heterogeneity since pre-surgery disease stages, and raising the combined NGS/dPCR sensitivity to 58.8%. ctDNA levels at surgery correlated with neither tumor size, stage, grade, or nodal status, nor with variant abundance in paired tDNA. LB sensitivity reached 63.6% when ctDNA was combined with CEA. Finally, persistence and absence of ctDNA on the first conventional (month 3) post-surgery follow-up were associated with fast relapse and a disease-free status in 3 and 7 patients, respectively.

Conclusions: A simple clinical NGS/dPCR/CEA combination effectively addresses the LB challenge in a fraction of non-metastatic CRC patients.
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http://dx.doi.org/10.1186/s13046-020-01569-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168847PMC
April 2020

MKK3 sustains cell proliferation and survival through p38DELTA MAPK activation in colorectal cancer.

Cell Death Dis 2019 11 6;10(11):842. Epub 2019 Nov 6.

Laboratory of Medical Physics and Expert Systems, Department of Diagnostic Research and Technological Innovation, IRCCS - Regina Elena National Cancer Institute, 00144 Rome, Italy.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and understanding its underlying molecular mechanisms is crucial for the development of therapeutic strategies. The mitogen-activated protein kinase-kinase 3 (MKK3) is a specific activator of p38 MAP kinases (p38 MAPKs), which contributes to the regulation of several cellular functions, such as proliferation, differentiation, apoptosis as well as response to drugs. At present, the exact MKK3/p38 MAPK pathway contribution in cancer is heavily debated because of its pleiotropic function. In this work, we retrospectively explored the prognostic and pathobiologic relevance of MKK3 in a cohort of CRC patients and assessed MKK3 molecular functions in a panel of CRC lines and colonocytes primary cultures. We found increased MKK3 levels in late-stage CRC patients which correlated with shorter overall survival. Herein, we report that the MKK3 targeting by inducible RNA interference univocally exerts antitumor effects in CRC lines but not in primary colonocytes. While MKK3 depletion per se affects growth and survival by induction of sustained autophagy and death in some CRC lines, it potentiates response to chemotherapeutic drug 5-fluorouracil (5-FU) in all of the tested CRC lines in vitro. Here, we demonstrate for the first time that in CRC the MKK3 specifically activates p38delta MAPK isoform to sustain prosurvival signaling and that such effect is exacerbated upon 5-FU challenge. Indeed, p38delta MAPK silencing recapitulates MKK3 depletion effects in CRC cells in vitro and in vivo. Overall, our data identified a molecular mechanism through which MKK3 supports proliferation and survival signaling in CRC, further supporting MKK3 as a novel and extremely attractive therapeutic target for the development of promising strategies for the management of CRC patients.
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http://dx.doi.org/10.1038/s41419-019-2083-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834673PMC
November 2019

TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment.

Nucleic Acids Res 2019 04;47(7):3365-3382

Oncogenomic and Epigenetic Unit, IRCCS - Regina Elena National Cancer Institute, Via Elio Chianesi 53, Rome 00144, Italy.

The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.
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http://dx.doi.org/10.1093/nar/gkz041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468246PMC
April 2019

JAK/Stat5-mediated subtype-specific lymphocyte antigen 6 complex, locus G6D (LY6G6D) expression drives mismatch repair proficient colorectal cancer.

J Exp Clin Cancer Res 2019 Jan 22;38(1):28. Epub 2019 Jan 22.

Department of Sciences and Technologies, University of Sannio, Via Port'Arsa, 1182100, Benevento, Italy.

Background: Human microsatellite-stable (MSS) colorectal cancers (CRCs) are immunologically "cold" tumour subtypes characterized by reduced immune cytotoxicity. The molecular linkages between immune-resistance and human MSS CRC is not clear.

Methods: We used transcriptome profiling, in silico analysis, immunohistochemistry, western blot, RT-qPCR and immunofluorescence staining to characterize novel CRC immune biomarkers. The effects of selective antagonists were tested by in vitro assays of long term viability and analysis of kinase active forms using anti-phospho antibodies.

Results: We identified the lymphocyte antigen 6 complex, locus G6D (LY6G6D) as significantly overexpressed (around 15-fold) in CRC when compared with its relatively low expression in other human solid tumours. LY6G6D up-regulation was predominant in MSS CRCs characterized by an enrichment of immune suppressive regulatory T-cells and a limited repertoire of PD-1/PD-L1 immune checkpoint receptors. Coexpression of LY6G6D and CD15 increases the risk of metastatic relapse in response to therapy. Both JAK-STAT5 and RAS-MEK-ERK cascades act in concert as key regulators of LY6G6D and Fucosyltransferase 4 (FUT4), which direct CD15-mediated immune-resistance. Momelotinib, an inhibitor of JAK1/JAK2, consistently abrogated the STAT5/LY6G6D axis in vitro, sensitizing MSS cancer cells with an intact JAK-STAT signaling, to efficiently respond to trametinib, a MEK inhibitor used in clinical setting. Notably, colon cancer cells can evade JAK2/JAK1-targeted therapy by a reversible shift of the RAS-MEK-ERK pathway activity, which explains the treatment failure of JAK1/2 inhibitors in refractory CRC.

Conclusions: Combined targeting of STAT5 and MAPK pathways has superior therapeutic effects on immune resistance. In addition, the new identified LY6G6D antigen is a promising molecular target for human MSS CRC.
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http://dx.doi.org/10.1186/s13046-018-1019-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343337PMC
January 2019

The clinical significance of PD-L1 in advanced gastric cancer is dependent on mutations and ATM expression.

Oncoimmunology 2018;7(8):e1457602. Epub 2018 Apr 24.

Division of Medical Oncology 2, "Regina Elena" National Cancer Institute, Via Elio Chianesi 53, Rome, Italy.

Whether PD-L1 expression is associated with survival outcomes in gastric cancer (GC) is controversial. The inhibition of the PD-1/PD-L1 pathway is effective against genomically unstable tumors. Hypothesizing that also the clinical significance of PD-L1 might be dependent on the activation of molecular circuits ensuring genomic stability, we evaluated PD-L1 expression in tissue samples from 72 advanced GC patients treated with first-line chemotherapy. Samples were already characterized for DNA damage repair (DDR) component expression (pATM, pChk1, pWee1, γ-H2AX and pRPA2) along with mutations in DDR-linked genes ( and ). Overall, PD-L1 expression was not associated with progression-free survival (PFS) and overall survival (OS), independently on whether we considered its expression in tumor cells (PD-L1-TCs) or in the immune infiltrate (PD-L1-TILs). In subgroup analysis, positive PD-L1-TC immunostaining was associated with better PFS in patients whose tumors did not carry DDR activation (multivariate Cox: HR 0.34, 95%CI: 0.15-0.76, p = 0.008). This subset (DDR) was characterized by negative pATM expression or the presence of mutations. Conversely, the relationship between PD-L1-TC expression and PFS was lost in a molecular scenario denoting DDR activation (DDR), as defined by concomitant pATM expression and wild-type form. Surprisingly, while PD-L1-TC expression was associated with better OS in the DDR subset (multivariate Cox: HR 0.41, 95% CI: 0.17-0.96, p = 0.039), in the DDR subgroup we observed an opposite impact on OS (multivariate Cox: HR 2.56, 95%CI: 1.06-6.16, p = 0.036). Thus, PD-L1-TC expression may impact survival outcomes in GC on the basis of the activation/inactivation of genome-safeguarding pathways.
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http://dx.doi.org/10.1080/2162402X.2018.1457602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136851PMC
April 2018

Coexisting YAP expression and TP53 missense mutations delineates a molecular scenario unexpectedly associated with better survival outcomes in advanced gastric cancer.

J Transl Med 2018 09 4;16(1):247. Epub 2018 Sep 4.

Division of Medical Oncology 2, IRCCS "Regina Elena" National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

We have previously reported that nuclear expression of the Hippo transducer TAZ in association with Wnt pathway mutations negatively impacts survival outcomes in advanced gastric cancer (GC) patients. Here, we extended these previous findings by investigating another oncogenic cooperation, namely, the interplay between YAP, the TAZ paralogue, and p53. The molecular output of the YAP-p53 cooperation is dependent on TP53 mutational status. In the absence of mutations, the YAP-p53 crosstalk elicits a pro-apoptotic response, whereas in the presence of TP53 mutations it activates a pro-proliferative transcriptional program. In order to study this phenomenon, we re-analyzed data from 83 advanced GC patients treated with chemotherapy whose tissue samples had been characterized for YAP expression (immunohistochemistry, IHC) and TP53 mutations (deep sequencing). In doing so, we generated a molecular model combining nuclear YAP expression in association with TP53 missense variants (YAP+/TP53). Surprisingly, this signature was associated with a decreased risk of disease progression (multivariate Cox for progression-free survival: HR 0.53, 95% CI 0.30-0.91, p = 0.022). The YAP+/TP53 model was also associated with better OS in the subgroup of patients who received chemotherapy beyond the first-line setting (multivariate Cox: HR 0.36, 95% CI 0.16-0.81, p = 0.013). Collectively, our findings suggest that the oncogenic cooperation between YAP and mutant p53 may translate into better survival outcomes. This apparent paradox can be explained by the pro-proliferative program triggered by YAP and mutant p53, that supposedly renders cancer cells more vulnerable to cytotoxic therapies.
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http://dx.doi.org/10.1186/s12967-018-1607-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122687PMC
September 2018

Deep sequencing and pathway-focused analysis revealed multigene oncodriver signatures predicting survival outcomes in advanced colorectal cancer.

Oncogenesis 2018 Jul 22;7(7):55. Epub 2018 Jul 22.

Division of Medical Oncology 2, IRCCS "Regina Elena" National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Genomic technologies are reshaping the molecular landscape of colorectal cancer (CRC), revealing that oncogenic driver mutations (APC and TP53) coexist with still underappreciated genetic events. We hypothesized that mutational analysis of CRC-linked genes may provide novel information on the connection between genetically-deregulated pathways and clinical outcomes. We performed next-generation sequencing (NGS) analysis of 16 recurrently mutated genes in CRC exploiting tissue specimens from 98 advanced CRC patients. Multiple correspondence analysis (MCA) was used to identify gene sets characterizing negative and positive outliers (patients in the lowest and highest quartile of progression-free survival, PFS). Variables potentially affecting PFS and overall survival (OS) were tested in univariate and multivariate Cox proportional hazard models. Sensitivity analyses and resampling were used to assess the robustness of genomic predictors. MCA revealed that APC and TP53 mutations were close to the negative outlier group, whereas mutations in other WNT pathway genes were in proximity of the positive outliers. Reasoning that genetic alterations interact epistatically, producing greater or weaker consequences in combination than when individually considered, we tested whether patients whose tumors carried a genetic background characterized by APC and TP53 mutations without coexisting mutations in other WNT genes (AMER1, FBXW7, TCF7L2, CTNNB1, SOX9) had adverse survival outcomes. With this approach, we identified two oncodriver signatures (ODS1 and ODS2) associated with shorter PFS (ODS1 multivariate Cox for PFS: HR 2.16, 95%CI: 1.28-3.64, p = 0.004; ODS2 multivariate Cox for PFS: HR 2.61, 95%CI: 1.49-4.58, p = 0.001). Clinically-focused and molecularly-focused sensitivity analyses, resampling, and reclassification of mutations confirmed the stability of ODS1/2. Moreover, ODS1/2 negatively impacted OS. Collectively, our results point to co-occurring driver mutations as an adverse molecular factor in advanced CRC. This relationship depends on a broader genetic context highlighting the importance of genetic interactions.
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http://dx.doi.org/10.1038/s41389-018-0066-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054833PMC
July 2018

HMGA1/E2F1 axis and NFkB pathways regulate LPS progression and trabectedin resistance.

Oncogene 2018 11 6;37(45):5926-5938. Epub 2018 Jul 6.

Cellular Network and Molecular Therapeutic Target Unit, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144, Roma, Italy.

Although the medical treatments of sarcoma have evolved in the last years, a significant portion of patients develops recurrence after therapies suggesting the need to identify novel targets to improve the treatments. By the use of patient-derived and established cell lines from liposarcoma, as well as specimens from patient biopsies, we found that HMGA1 is involved in the progression of dedifferentiated and myxoid liposarcoma. The immunohistochemical and RT-PCR analyses of 68 liposarcoma specimens revealed a significant high expression of HMGA1, at the protein and RNA levels, both in myxoid and dedifferentiated liposarcoma subtypes compared with differentiated ones. Loss- and gain-of-function experiments by HMGA1-specific depletion and overexpression in dedifferentiated and myxoid liposarcoma cells showed the contribution of this oncogenic factor in cell proliferation, motility, invasion, and drug resistance. The in vitro and in vivo treatment of myxoid liposarcoma with trabectedin, a drug with a potent anti-tumor activity, revealed downregulation of HMGA1, E2F1, and its-downstream targets, vimentin and ZEB1, indicating a critical role of trabectedin in inhibiting the mesenchymal markers of these tumors through the HMGA1/E2F1 axis. These data were also confirmed in patients' tumor biopsies being HMGA1, E2F1, and vimentin expression significantly reduced upon trabectedin therapy, administered as neo-adjuvant chemotherapy. Furthermore, trabectedin treatment inhibits in vitro NFkB pathway in mixoyd liposarcoma sensitive but not in resistant counterparts, and the inhibition of NFkB pathway re-sensitizes the resistant cells to trabectedin treatment. These data support the rational for combining NFkB inhibitors with trabectedin in liposarcoma patients, who have become resistant to the drug.
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http://dx.doi.org/10.1038/s41388-018-0394-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224401PMC
November 2018

Liquid biopsy in mice bearing colorectal carcinoma xenografts: gateways regulating the levels of circulating tumor DNA (ctDNA) and miRNA (ctmiRNA).

J Exp Clin Cancer Res 2018 Jun 26;37(1):124. Epub 2018 Jun 26.

Department of Life Sciences and Biotechnology, Biochemistry and Molecular Biology Section, Ferrara University, Via Fossato di Mortara 74, 44121, Ferrara, Italy.

Background: Circulating tumor DNA (ctDNA) and miRNA (ctmiRNA) are promising biomarkers for early tumor diagnosis, prognosis and monitoring, and to predict therapeutic response. However, a clear understanding of the fine control on their circulating levels is still lacking.

Methods: Three human colorectal carcinoma cell lines were grown in culture and as tumor xenograft models in nude mice. Chip-based and droplet digital PCR platforms were used to systematically and quantitatively assess the levels of DNAs and miRNAs released into the culture supernatants and mouse blood plasma.

Results: Strikingly, mutated DNAs from the same (KRAS) and different (PIK3CA and FBWX7) genomic loci were differentially detected in culture supernatants and blood, with LS174T releasing 25 to 60 times less DNA in culture, but giving rise to 7 to 8 times more DNA in blood than LoVo cells. Greater LS174T ctDNA accumulation occurred in spite of similar CD31 immunostaining (micro-vascularization) and lesser proliferation and tissue necrosis as compared to LoVo. As to the three selected miRNAs (miR-221, miR-222 and miR-141), all of them were constitutively present in the plasma of tumor-free mice. Micro-RNA miR-141 was released into HT-29 cell supernatants 10 and 6.5 times less abundantly with respect to LoVo and LS174T, respectively; on the contrary, release of miR-141 in blood of HT-29 xenografted mice was found similar to that observed in LoVo and LS174T mice.

Conclusions: Taken together, our results support the existence of multiple, finely tuned (non-housekeeping) control gateways that selectively regulate the release/accumulation of distinct ctDNA and miRNA species in culture and tumor xenograft models. Different xenografts (proxies of different patients) considerably differ in gateway usage, adding several layers of complexity to the well-known idea of molecular heterogeneity. We predict that even high tissue representation of mutated DNA and miRNA may result in insufficient diagnostic analyte representation in blood. In this respect, our data show that careful modeling in mice may considerably help to alleviate complexity, for instance by pre-screening for the most abundant circulating analytes in enlarged sets of tumor xenografts.
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http://dx.doi.org/10.1186/s13046-018-0788-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020232PMC
June 2018

Expression of the Hippo transducer TAZ in association with WNT pathway mutations impacts survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy.

J Transl Med 2018 02 5;16(1):22. Epub 2018 Feb 5.

Division of Medical Oncology 2, "Regina Elena" National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Background: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy.

Methods: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS).

Results: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZ/WNT) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZ/WNT signature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZ/WNT signature negatively impacted overall survival.

Conclusions: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes.
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http://dx.doi.org/10.1186/s12967-018-1385-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800016PMC
February 2018

HSP90 inhibition alters the chemotherapy-driven rearrangement of the oncogenic secretome.

Oncogene 2018 03 9;37(10):1369-1385. Epub 2018 Jan 9.

Oncogenomic and Epigenetic Unit Regina Elena National Cancer Institute, Rome, Italy.

Adaptive resistance to therapy is a hallmark of cancer progression. To date, it is not entirely clear how microenvironmental stimuli would mediate emergence of therapy-resistant cell subpopulations, although a rearrangement of the cancer cell secretome following therapy-induced stress can be pivotal for such a process. Here, by using the highly chemoresistant malignant pleural mesothelioma (MPM) as an experimental model, we unveiled a key contribution of the chaperone HSP90 at assisting a chemotherapy-instigated Senescence-Associated-Secretory-Phenotype (SASP). Thus, administration of a clinical trial grade, HSP90, inhibitor blunted the release of several cytokines by the chemotherapy-treated MPM cells, including interleukin (IL)-8. Reduction of IL-8 levels hampered the FAK-AKT signaling and inhibited 3D growth and migration. This correlated with downregulation of key EMT and chemoresistance genes and affected the survival of chemoresistant ALDH cell subpopulations. Altogether, inhibition of HSP90 provoked a switch from a pro-tumorigenic SASP to a pro-apoptotic senescence status, thus resulting in chemosensitizing effects. In mouse xenografts treated with first-line agents, inhibiting HSP90 blunted FAK activation and reduced the expression of ALDH1A3 and the levels of circulating human IL-8, these latter strongly correlating with the effect on tumor growth. We validated the above findings in primary mesothelioma cultures, a more clinically relevant model. We unveiled here a key contribution of the chaperone HSP90 at assisting the secretory stress in chemotherapy-treated cells, which may warrant further investigation in combinatorial therapeutic settings.
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http://dx.doi.org/10.1038/s41388-017-0044-8DOI Listing
March 2018

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells.

Gut 2018 05 7;67(5):903-917. Epub 2017 Apr 7.

Institute of General Pathology, Catholic University and A. Gemelli Polyclinic, Rome, Italy.

Objective: Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.

Design: To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.

Results: The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.

Conclusions: LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.
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http://dx.doi.org/10.1136/gutjnl-2016-312623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890648PMC
May 2018

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy.

Int J Cancer 2017 06 11;140(11):2587-2595. Epub 2017 Mar 11.

Division of Medical Oncology 2, "Regina Elena" National Cancer Institute, Rome, Italy.

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ-H2AX /pATM ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the γ-H2AX /pATM model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The γ-H2AX /pATM model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance.
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http://dx.doi.org/10.1002/ijc.30668DOI Listing
June 2017

SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage.

Nucleic Acids Res 2017 02;45(4):1820-1834

Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Via Elio Chianesi 53, Rome 00144, Italy.

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.
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http://dx.doi.org/10.1093/nar/gkw1202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389694PMC
February 2017

Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers.

Oncotarget 2017 Dec 19;8(65):108463-108479. Epub 2016 Aug 19.

Department of Research, Advanced Diagnostic, and Technological Innovation, IRCCS Regina Elena National Cancer Institute, Rome, Italy.

Although the medical treatment of colorectal cancer has evolved greatly in the last years, a significant portion of early-stage patients develops recurrence after therapies. The current clinical trials are directed to evaluate new drug combinations and treatment schedules. By the use of patient-derived or established colon cancer cell lines, we found that the tyrosine kinase receptor HER3 is involved in the mechanisms of resistance to therapies. In agreement, the immunohistochemical analysis of total and phospho-HER3 expression in 185 colorectal cancer specimens revealed a significant correlation with lower disease-free survival. Targeting HER3 by the use of the monoclonal antibody patritumab we found induction of growth arrest in all cell lines. Despite the high efficiency of patritumab in abrogating the HER3-dependent activation of PI3K pathway, the HER2 and EGFR-dependent MAPK pathway is activated as a compensatory mechanism. Interestingly, we found that the MEK-inhibitor trametinib inhibits, as expected, the MAPK pathway but induces the HER3-dependent activation of PI3K pathway. The combined treatment results in the abrogation of both PI3K and MAPK pathways and in a significant reduction of cell proliferation and survival. These data suggest a new strategy of therapy for HER3-overexpressing colon cancers.
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http://dx.doi.org/10.18632/oncotarget.11400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752456PMC
December 2017

BCAM and LAMA5 Mediate the Recognition between Tumor Cells and the Endothelium in the Metastatic Spreading of KRAS-Mutant Colorectal Cancer.

Clin Cancer Res 2016 Oct 3;22(19):4923-4933. Epub 2016 May 3.

Candiolo Cancer Institute-FPO, IRCCS, Candiolo (Turin), Italy. Department of Oncology, University of Turin, Candiolo (Turin), Italy. University of New Mexico Comprehensive Cancer Center. Albuquerque, New Mexico. Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico.

Purpose: KRAS mutations confer adverse prognosis to colorectal cancer, and no targeted therapies have shown efficacy in this patient subset. Paracrine, nongenetic events induced by KRAS-mutant tumor cells are expected to result in specific deregulation and/or relocation of tumor microenvironment (TME) proteins, which in principle can be exploited as alternative therapeutic targets.

Experimental Design: A multimodal strategy combining ex vivo/in vitro phage display screens with deep-sequencing and bioinformatics was applied to uncover TME-specific targets in KRAS-mutant hepatic metastasis from colorectal cancer. Expression and localization of BCAM and LAMA5 were validated by immunohistochemistry in preclinical models of human hepatic metastasis and in a panel of human specimens (n = 71). The antimetastatic efficacy of two BCAM-mimic peptides was evaluated in mouse models. The role of BCAM in the interaction of KRAS-mutant colorectal cancer cells with TME cells was investigated by adhesion assays.

Results: BCAM and LAMA5 were identified as molecular targets within both tumor cells and TME of KRAS-mutant hepatic metastasis from colorectal cancer, where they were specifically overexpressed. Two BCAM-mimic peptides inhibited KRAS-mutant hepatic metastasis in preclinical models. Genetic suppression and biochemical inhibition of either BCAM or LAMA5 impaired adhesion of KRAS-mutant colorectal cancer cells specifically to endothelial cells, whereas adhesion to pericytes and hepatocytes was unaffected.

Conclusions: These data show that the BCAM/LAMA5 system plays a functional role in the metastatic spreading of KRAS-mutant colorectal cancer by mediating tumor-TME interactions and as such represents a valuable therapeutic candidate for this large, currently untreatable patient group. Clin Cancer Res; 22(19); 4923-33. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2664DOI Listing
October 2016

Cancer Stem Cell-Based Models of Colorectal Cancer Reveal Molecular Determinants of Therapy Resistance.

Stem Cells Transl Med 2016 Apr 8;5(4):511-23. Epub 2016 Mar 8.

Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

Unlabelled: Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]-targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)-driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC-enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC-enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti-EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance.

Significance: Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR-targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.
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http://dx.doi.org/10.5966/sctm.2015-0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798739PMC
April 2016

Role of gonadotropin-releasing hormone analogues in metastatic male breast cancer: results from a pooled analysis.

J Hematol Oncol 2015 May 17;8:53. Epub 2015 May 17.

Division of Medical Oncology B, "Regina Elena" National Cancer Institute, Via Elio Chianesi 53, 00144, Rome, Italy.

Background: Male breast cancer is a rare malignancy. Despite the lack of prospectively generated data from trials in either the adjuvant or metastatic setting, patients are commonly treated with hormone therapies. Much controversy exists over the use of gonadotropin-releasing hormone analogues in metastatic male breast cancer patients. We conducted this study to provide more concrete ground on the use of gonadotropin-releasing hormone analogues in this setting.

Methods: We herein present results from a pooled analysis including 60 metastatic male breast cancer patients treated with either an aromatase inhibitor or cyproterone acetate as a monotherapy (23 patients) or combined with a gonadotropin-releasing hormone analogue (37 patients).

Results: Overall response rate was 43.5% in patients treated with monotherapy and 51.3% with combination therapy (p = 0.6). Survival outcomes favored combination therapy in terms of median progression-free survival (11.6 months versus 6 months; p = 0.05), 1-year progression-free survival rate (43.2% versus 21.7%; p = 0.05), median overall survival (29.7 months versus 22 months; p = 0.05), and 2-year survival rate (64.9% versus 43.5%; p = 0.05).

Conclusions: In metastatic male breast cancer patients, the combined use of gonadotropin-releasing hormone analogues and aromatase inhibitors or antiandrogens seems to be associated with greater efficacy, particularly in terms of survival outcomes, compared with monotherapy. Collectively, these results encourage considering these agents in the metastatic setting.
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http://dx.doi.org/10.1186/s13045-015-0147-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440498PMC
May 2015

Cancer stem cells: are they responsible for treatment failure?

Future Oncol 2014 Oct;10(13):2033-44

Division of Medical Oncology B, 'Regina Elena' National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy.

Overcoming resistance to standard anticancer treatments represents a significant challenge. The interest regarding cancer stem cells, a cellular population that has the ability to self-renew and to propagate the tumor, was prompted by experimental evidence delineating the molecular mechanisms that are selectively activated in this cellular subset in order to survive chemotherapy. This has also stimulated combination strategies aimed at rendering cancer stem cells vulnerable to anticancer agents. Moreover, cancer stem cells offer a unique opportunity for modeling human cancers in mice, thus emerging as a powerful tool for testing novel drugs and combinations in a simulation of human disease. These novel animal models may lay the foundation for a new generation of clinical trials aimed at anticipating the benefit to patients of anticancer therapies.
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http://dx.doi.org/10.2217/fon.14.126DOI Listing
October 2014