Cristina Ghirelli

Role of HER2 in wound-induced breast carcinoma proliferation

OBJECTIVE Clinical and experimental data have suggested that surgical removal of primary tumours promotes the growth of metastatic lesions. We assessed the effect of surgery on proliferation of breast carcinomas, in particular those overexpressing HER2 oncoprotein. METHODS Proliferation of breast carcinoma cells was assessed by MIB-1 immunohistochemistry in sections of primary breast carcinomas and in residual tumour found in re-excision specimens, and in in-vitro cell lines by colorimetric assay. Epidermal growth factor (EGF)-like growth factors were measured by displacement of radiolabelled EGF from its receptor. Cellular damage was measured in terms of creatine phosphokinase level. Downmodulation of HER2 was investigated by cytoplasmic expression of anti-HER2 antibody and by inhibition with anti-HER2 antibody trastuzumab. FINDINGS Residual breast carcinomas that had been surgically removed within 48 days after first surgery showed a significant increase in proliferation if they were HER2-positive. Wound drainage fluid and postsurgical serum samples from patients stimulated in-vitro growth of HER2-overexpressing breast carcinoma cells. Removal of HER2 from the cell membrane led to a striking reduction of the induced proliferation. The amount of EGF-like growth factors in post-surgical serum samples, as well as the extent of drainage-fluid-induced proliferation, directly correlated with the amount of surgical damage assessed by creatine phosphokinase levels (r=0.77, p=0.002 and r=0.69, p=0.009, respectively). Treatment of HER2-positive tumour cells with trastuzumab before adding the growth stimulus abolished drainage-fluid-induced proliferation. INTERPRETATION HER2 overexpression by breast carcinoma cells has a role in postsurgery stimulation of growth of breast carcinoma cells.

Formation of the 67-kDa laminin receptor by acylation of the precursor

Even though the involvement of the 67‐kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full‐length gene encoding a 37‐kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67‐kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120‐kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP‐40‐lysis buffer whereas the 37LRP is NP‐40‐insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP‐40‐soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67‐ and the 120‐kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross‐reacting molecule. J. Cell. Biochem. 69:244–251, 1998.

PDGFRβ and FGFR2 mediate endothelial cell differentiation capability of triple negative breast carcinoma cells

Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes.

Maspin influences response to doxorubicin by changing the tumor microenvironment organization

Altered degradation and deposition of extracellular matrix are hallmarks of tumor progression and response to therapy. From a microarray supervised analysis on a dataset of chemotherapy‐treated breast carcinoma patients, maspin, a member of the serpin protease inhibitor family, has been the foremost variable identified in non‐responsive versus responsive tumors. Accordingly, in a series of 52 human breast carcinomas, we detected high maspin expression in tumors that progressed under doxorubicin (DXR)‐based chemotherapy. Our analysis of the role of maspin in response to chemotherapy in human MCF7 and MDAMB231 breast and SKOV3 ovarian carcinoma cells transfected to overexpress maspin and injected into mice showed that maspin overexpression led to DXR resistance through the maspin‐induced collagen‐enriched microenvironment and that an anti‐maspin neutralizing monoclonal antibody reversed the collagen‐dependent DXR resistance. Impaired diffusion and decreased DXR activity were also found in tumors derived from Matrigel‐embedded cells, where abundant collagen fibers characterize the tumor matrix. Conversely, liposome‐based DXR reached maspin‐overexpressing tumor cells despite the abundant extracellular matrix and was more efficient in reducing tumor growth. Our results identify maspin‐induced accumulation of collagen fibers as a cause of disease progression under DXR chemotherapy for breast cancer. Use of a more hydrophilic DXR formulation or of a maspin inhibitor in combination with chemotherapy holds the promise of more consistent responses to maspin‐overexpressing tumors and dense‐matrix tumors in general.

Two novel monoclonal antibodies against the MUC4 tandem repeat reacting with an antigen overexpressed by lung cancer.

In this study we investigated the immunochemical and cytochemical reactivity of two monoclonal antibodies against the 16-amino acid tandem repeat of MUC4 to demonstrate a possible variation of the mucin core peptide expression related to lung cancer. The immunocytochemical anti-MUC4 reactivity was analyzed in four lung cancer cell lines (Calu-1, Calu-3, H460, SKMES) and in other tumor cell lines, as well as in frozen materials from 21 lung adenocarcinomas (ACs), including five bronchioloalveolar carcinomas (BACs), and 11 squamous cell lung carcinomas (SqCCs). A weak fluorescence anti-MUC4 positivity (range: 10.3–16.2) was observed only in acetone-fixed lung cancer cell lines Calu-1, Calu-3 and H460. These three lung cancer cell lines also showed a cytoplasmic immunoperoxidase reactivity. The immunostaining in lung cancer tissues showed a granular cytoplasmic reactivity: 15/21 (71%) and 17/21 (80%) ACs were positive with BC-LuC18.2 and BC-LuCF12, respectively. All BACs were positive. Moderate to strong reactivity was present in well-differentiated ACs. In the normal lung parenchyma counterparts weak reactivity was found only in bronchiolar cells. All SqCCs were negative. Anti-MUC4 reactivity was also observed in the alveolar mucus. In conclusion, our anti-MUC4 MAbs detect a secretion product present in mucus and this product is elaborated by lung cancer cells and overexpressed in well-differentiated lung ACs.

Production of a monoclonal antibody directed against the high-affinity nerve growth factor receptor.

The high-affinity nerve growth factor receptor corresponds to the tyrosine protein kinase encoded by the proto-oncogene trkA. Different findings suggest that nerve growth factor (NGF) can be operative in the growth modulation of tumor cell lines possessing high-affinity binding sites for this molecule. Using as immunizing material the SKNBE neuroblastoma cell line transfected with proto-trkA we produced a monoclonal antibody (MAb) able to recognize the high-affinity nerve growth factor receptor. The selected MAb, designated MGR12, is directed against an epitope present on the extracellular domain of the receptor since it showed reactivity on living trkA-expressing cells and was able to immunoprecipitate the proto-trkA molecule. The MGR12 MAb is directed against a non-functional epitope since it neither inhibited NGF binding nor induced receptor internalization. This new reagent appears to be an appropriate tool for analyzing the expression of high-affinity nerve growth factor receptor in tumors of different origin and for elucidating its involvement in tumor progression.

Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of Breast Cancer Cells

The tumor‐suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho‐MAPK, and phospho‐STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation. J. Cell. Physiol. 9999: 2661–2670, 2015.

HER2 signaling regulates the tumor immune microenvironment and trastuzumab efficacy

ABSTRACT Through whole-transcriptome profiling of HER2+ breast carcinomas (BCs), we previously showed that those sensitive to trastuzumab are addicted to this oncoprotein and are enriched in immune pathways, raising the hypothesis that HER2 itself regulates immune cell recruitment. In the present study we investigated the relationship between HER2 activity and the pro-trastuzumab tumor immune milieu. Gene expression profiling and immunohistochemistry analysis of 53 HER2+ BCs showed that trastuzumab-sensitive tumors expressed significantly higher levels of chemokines involved in immune cell recruitment, with higher infiltration of T cells and monocytes, and higher levels of PD-1 ligands than tumors that do not benefit from trastuzumab. In vitro analysis in HER2+ BC cells revealed that CCL2 production was induced by HER2 stimulation with EGF/HRG via the PI3K-NF-kB axis, and down-modulated by HER2 inhibition with trastuzumab. CCL2 expression was higher in HER2+/ER− than HER2+/ER+ BC cell lines, and degradation of ER by fulvestrant induced an enhancement in NF-κB transcriptional activity and consequent CCL2 expression. Trastuzumab efficacy relied on CCL2 levels and monocytes present in the tumor microenvironment in FVB mice bearing HER2+ mammary carcinoma cells. HER2 signals were also found to sustain the expression of PD-1 ligands in tumor cells via the MEK pathway. Overall, our results support the concept that the activated HER2 oncogene regulates recruitment and activation of tumor infiltrating immune cells and trastuzumab activity by inducing CCL2 and PD-1 ligands and that ER activity negatively controls the HER2-driven pro-trastuzumab tumor microenvironment.

The PDGFRβ/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer

BackgroundCDCP1, a transmembrane protein with tumor pro-metastatic activity, was recently identified as a prognostic marker in TNBC, the most aggressive breast cancer subtype still lacking an effective molecular targeted therapy. The mechanisms driving CDCP1 over-expression are not fully understood, although several stimuli derived from tumor microenvironment, such as factors present in Wound Healing Fluids (WHFs), reportedly increase CDCP1 levels.MethodsThe expression of CDCP1, PDGFRβ and ERK1/2cell was tested by Western blot after stimulation of MDA-MB-231 cells with PDGF-BB and, similarly, in presence or not of ERK1/2 inhibitor in a panel of TNBC cell lines. Knock-down of PDGFRβ was established in MDA-MB-231 cells to detect CDCP1 upon WHF treatment. Immunohistochemical staining was used to detect the expression of CDCP1 and PDGFRβ in TNBC clinical samples.ResultsWe discovered that PDGF-BB-mediated activation of PDGFRβ increases CDCP1 protein expression through the downstream activation of ERK1/2. Inhibition of ERK1/2 activity reduced per se CDCP1 expression, evidence strengthening its role in CDCP1 expression regulation. Knock-down of PDGFRβ in TNBC cells impaired CDCP1 increase induced by WHF treatment, highlighting the role if this receptor as a central player of the WHF-mediated CDCP1 induction. A significant association between CDCP1 and PDGFRβ immunohistochemical staining was observed in TNBC specimens, independently of CDCP1 gene gain, thus corroborating the relevance of the PDGF-BB/PDGFRβ axis in the modulation of CDCP1 expression.ConclusionWe have identified PDGF-BB/PDGFRβ–mediated pathway as a novel player in the regulation of CDCP1 in TNCBs through ERK1/2 activation. Our results provide the basis for the potential use of PDGFRβ and ERK1/2 inhibitors in targeting the aggressive features of CDCP1-positive TNBCs.

Abstract 4123: CRISPR-Cas9 and siRNA screening in primary human immune cells

A major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumour responses. Screens using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-mediated genome editing seem well placed to identify new targets. However, although CRISPR-Cas9 gene editing works well in primary T cells using electroporation, use of a lentivirus one vector system has proved challenging in primary T cells compared with cancer cell lines. We have used several different approaches to identify the most useful method for transduction of primary human T cells with CRISPR components. Electroporation of sgRNAs and mRNA encoding Cas9 into proliferating T cells efficiently generate T cells with specific gene knock-outs or knock-ins, with targeting rates of around 37% for gene knockout. Thus, primary T cells are amenable to CRISPR-Cas9 gene editing, and the capacity to rapidly modify loci enables generation of primary T cell models suitable for comprehending the function of modified receptor-ligand pairs involved in an immune checkpoint response. Our pooled sgRNA-Cas9 screens in cancer cell lines have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing. However, use of the same approach in primary T cells has not resulted in efficient transduction of the library. Specifically, isolated CD3+ T cells stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2 resulted in no expression or low level expression of GFP after cells were transduced with a one vector CRISPR-Cas9 sgRNA library. Our experiments indicate, in line with published data, that T cells can be transduced effectively with lentivirus, thus we are examining the use of a two vector CRISPR-Cas9 system and the use of CRISPRi to idealise CRISPR screening in primary T cells. We are also carrying out target identification and validation in myeloid derived suppressor cells (MDSCs). We are using an siRNA approach in these cells, which are generated by PBMC co-culture with cancer cell lines for 7 days, or by culture in the presence of recombinant GM-CSF and IL-6 for 7 days. Our initial data indicate that these MDSCs can effectively suppress autologous, as well as allogeneic, CD8 T cell proliferation mediated by anti-CD3 and anti-CD28 stimulation and that siRNA knockdown is effective in MDSCs. We will use our druggable genome plus arrayed siRNA library to identify targets that when knocked down inhibit the capacity of MDSCs to suppress T cell proliferation. We anticipate that these data will be useful in identifying new targets that are involved in regulating an immune response to tumour development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Simon Scrace, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 and siRNA screening in primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2017-4123