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Background The improved immunological understanding revealed the tumor microenvironment as an appealing driver to restore the immune response against cancer cells resulting in a paradigm shift in the oncology field. However, the complexity of the tumor milieu suggests a role of several pathways linking in immunomodulation mechanisms. Pancreatic cancer represents a model of the intricate relationship between malignant cells and their surrounding neighborhood. Research design and methods In this study we analyzed, retrospectively, 6 cases of rare pancreatic sarcomatoid carcinoma (PSC) and evaluated the expression of PD-L1 and Notch, aiming to explore new attributes in immunophenotype. Results PD-L1 CPS≥1% was common in PSCs (83%) with half samples expressing PD-L1 CPS≥50%. Notch1 and Notch3 expression resulted positive demonstrating a high IRS range of expression. A direct significant correlation between PD-L1 and Nocth3 overexpression (r=0.7; p=0.036) has been observed. Moreover, immunofluorescence studies revealed a co-localization of Notch3 and PD-L1 when both proteins were over-expressed within cytoplasmic or membranous compartments of the same cells. Conclusions Our data identify a unique biological characterization of this rare pancreatic histotype. These findings provide a rationale for future studies evaluating the potential crosstalk between PD-L1/PD-1 axis and Notch pathways and prompting the development of novel therapeutics strategy.

Mucosal high-risk (HR) human papillomaviruses (HPV) are associated with anogenital carcinogenesis. The products of two early genes, E6 and E7, act as major viral oncoproteins. Functional studies in experimental models showed that HPV16 E6 induces degradation of the PDZ protein, the Na+/H+ exchanger regulatory factor-1 (NHERF-1). Here, we determined NHERF-1 protein levels by immunohistochemistry (IHC) in (i) benign anogenital warts (n = 8) (ii) premalignant lesions (L-SIL and H-SIL) (n = 43) and (iii) invasive cervical squamous cell carcinomas (SCC) (n = 17). A decrease of NHERF-1 protein level was not observed in genital warts in comparison to healthy epithelium. Conversely, a clearly decrease in NHERF-1 protein levels was observed in HPV16-positive pre-malignant and malignant lesions, while the phenomenon was much attenuated in lesions induced by other HR HPV types. In conclusion, these findings show that mucosal HPV types differently impact on NHERF-1 protein level in benign and malignant anogenital lesions.

Undifferentiated sarcomatoid carcinoma (USC) of the pancreas is a rare but especially aggressive variant of pancreatic ductal adenocarcinoma (PDAC), composed of at least 80% of sarcomatoid cells. This study aimed to elucidate its clinicopathological and molecular features. The study cohort included 10 patients with pancreatic USC. Clinicopathological parameters were determined for each patient. The molecular profile was investigated using next-generation sequencing (NGS). Histologically, all tumors were hypercellular neoplasms with spindle-shaped or sarcomatoid cells. All patients showed vascular and perineural invasion. Most patients had a poor prognosis. NGS showed important similarities with conventional PDAC, including frequent alterations in the classic PDAC drivers, KRAS (100% of cases), TP53 (90%), and CDKN2A (60%). There were also some important distinctions from conventional PDAC: 1) SMAD4, a typical PDAC driver gene, was mutated in only one case (10%); 2) Another distinctive molecular feature was the recurrent KRAS amplification (30% of cases), which is very rare in conventional PDAC. It has been previously reported in another subtype of pancreatic un-differentiated carcinoma, the rhabdoid variant, and may be a key event leading to the acquisition of an undifferentiated phenotype in a subgroup of cases; 3) Lastly, in two different cases, we detected two potentially actionable targets, not belonging to the typical PDAC molecular landscape, such as MCL1 amplification and POLQ mutation. Our study sheds light on this rare tumor type, which shows aggressive biological behavior and few druggable alterations. The most distinctive molecular features of pancreatic USC are the paucity of SMAD4 alterations and recurrent KRAS amplification. (C) 2022 Elsevier Inc. All rights reserved.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease, and patients with apparently similar clinicopathological characteristics in clinical practice show different outcome. This study evaluated in primary BCs and in the subgroup of the triple-negative breast cancers (TNBCs) the level of tumor infiltrating lymphocytes (TILs), Na+/H+ exchanger regulatory factor 1 (NHERF1) expression, and their association respect to the clinical outcome of patients. MATERIAL AND METHODS: NHERF1 expression was assessed by immunohistochemistry in 338 BC samples; the analysis of TILs was examined using hematoxylin and eosin stained slides, according to International TILs Working Group 2014. RESULTS: Multivariate analysis identified TILs as an independent prognostic factor for DFS in the entire cohort and in the TNBC subgroup (HR, 0.32; 95% CI, 0.12–0.87; P = 0.026; and HR, 0.22; 95% CI, 0.06–0.80; P = 0.022, respectively). Univariate and survival analysis by Kaplan–Meier method revealed that patients with cytoplasmic (c) NHERF1-/TILs+ expression had better DFS than other patients (P = 0.049), and this result was also found in the TNBC subgroup (P = 0.031). Moreover, TNBC patients with cNHERF1−/TILs− expression had a worse DFS and OS than other patients (P = 0.057 and P = 0.002, respectively). CONCLUSIONS: In the complex scenario of BC and in the era of tumor immunogenicity and immunotherapy, we found an association of TIL levels and cNHERF1 expression that could be useful to identify BCs and particularly TNBC patients with different prognosis and clinical outcome.

Na + /H + exchanger regulatory factor 1 (NHERF1) is a scaffold protein, with two tandem PDZ domains and a carboxyl-terminal ezrin-binding (EB) region. This particular sticky structure is responsible for its interaction with different molecules to form multi-complexes that have a pivotal role in a lot of diseases. In particular, its involvement during carcinogenesis and cancer progression has been deeply analyzed in different tumors. The role of NHERF1 is not unique in cancer; its activity is connected to its subcellular localization. The literature data suggest that NHERF1 could be a new prognostic/predictive biomarker from breast cancer to hematological cancers. Furthermore, the high potential of this molecule as therapeutical target in different carcinomas is a new challenge for precision medicine. These evidences are part of a future view to improving patient clinical management, which should allow different tumor phenotypes to be treated with tailored therapies. This article reviews the biology of NHERF1, its engagement in different signal pathways and its involvement in different cancers, with a specific focus on breast cancer. It also considers NHERF1 potential role during inflammation related to most human cancers, designating new perspectives in the study of this "Janus-like" protein.

Tumor-infiltrating lymphocytes (TILs) are a valuable indicator of the immune microenvironment that plays the central role in new anticancer drugs. TILs have a strong prognostic role in triple negative breast cancer (TNBC). Little is known about the interaction with the androgen receptor (AR) and forkhead box A1 (FOXA1). We analyzed the relationships between TIL levels, AR, and FOXA1 expression and their clinical significance in TNBC patients. Further, we investigated their interaction with other biomarkers like programmed cell death ligand-1 (PD-L1), breast cancer type 1 susceptibility protein (BRCA1), poly (ADP-Ribose) polymerase 1 (PARP1), and Na+/H+ exchanger regulatory factor 1 (NHERF1). The expression of the proteins was evaluated by immunohistochemistry in 124 TNBC samples. TILs were performed adhering to International TILs Working Group 2014 criteria. Cox proportional hazards models were also used to identify risk factors associated with poor prognosis. Multivariate analysis identified TILs as independent prognostic factor of disease free survival (DFS, p = 0.045). A Kaplan&ndash, Meyer analysis revealed that the patients with high TILs had a better DFS compared to patients with low TILs (p = 0.037), and the phenotypes TILs&minus, /AR+ and TILs&minus, /FOXA1&minus, had a worse DFS (p = 0.032, p = 0.001 respectively). AR was associated with FOXA1 expression (p = 0.007), and the tumors FOXA1+ presented low levels of TILs (p = 0.028). A poor DFS was observed for AR+/FOXA1+ tumors compared to other TNBCs (p = 0.0117). Low TILs score was associated with poor patients&rsquo, survival, and TILs level in combination with AR or FOXA1 expression affected patient&rsquo, s clinical outcome. In addition, AR+/FOXA1+ phenotype identified a specific subgroup of TNBC patients with poor prognosis. These data may suggest new ways of therapeutic intervention to support current treatments.

Wnt pathway is involved in breast cancer (BC) progression. Our aim was to evaluate the expression of some components of the Wnt pathway (β-catenin, FZD4, LRP5, LRP6 and TCF1) in order to detect potential associations with NHERF1 protein. In addition, we assessed their impact on patients’clinical outcome. We evaluated 220 primary BC samples by immunohistochemistry (IHC) and protein localization by immunofluorescence. We found a significant correlation between NHERF1 and FZD4, LRP5, LRP6 and TCF1. Univariate analysis showed that β-catenin (p

International audience; Nuclear activated β-catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/β-catenin pathway mutations in APC/KRAS or β-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic β-catenin signaling negatively regulates the expression of NHERF1 (Na+/H+ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear β-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that β-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon β-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single β-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/β-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with β-catenin knockdown alone. Collectively, our data unveil novel β-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining β-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/β-catenin-targeted therapeutics.

The Wnt pathway is involved in the progression of breast cancer (BC). We aimed to evaluate the expression of some components of the Wnt pathway (&beta, catenin, FZD4 (frizzled receptor 4), LRP5 (low-density lipoprotein receptor-related protein 5), LRP6, and TCF1 (T-cell factor 1)) to detect potential associations with NHERF1 (Na+/H+ exchanger regulatory factor 1) protein. Besides, we assessed their impact on patients&rsquo, clinical outcome. We evaluated 220 primary BC samples by immunohistochemistry (IHC) and protein localization by immunofluorescence. We found a significant correlation between NHERF1 and FZD4, LRP5, LRP6, and TCF1. Univariate analysis showed that the overexpression of &beta, catenin (p &lt, 0.0001), FZD4 (p = 0.0001), LRP5, LRP6, and TCF1 (p &lt, 0.0001 respectively) was related to poor disease-free survival (DFS). A Kaplan-Meier analysis confirmed univariate data and showed a poor DFS for cNHERF1+/FZD4+ (p = 0.0007), cNHERF1+/LRP5+ (p = 0.0002), cNHERF1+/LRP6+ (p &lt, 0.0001), and cNHERF1+/TCF1+ phenotypes (p = 0.0034). In multivariate analysis, the expression of TCF1 and &beta, catenin was an independent prognostic variable of worse DFS (p = 0.009 and p = 0.027, respectively). In conclusion, we found that the overexpression of &beta, catenin, FZD4, LRP5, LRP6, and TCF1 was associated with poor prognosis. Furthermore, we first identified TCF1 as an independent prognostic factor of poor outcome, indicating it as a new potential biomarker for the management of BC patients. Also, the expression of Wnt pathway proteins, both alone and in association with NHERF1, suggests original associations of biological significance for new studies.

We designed 3-aroyl-1,4-diarylpyrrole (ARDAP) derivatives as potential anticancer agents having different\ud substituents at the 1- or 4-phenyl ring. ARDAP compounds exhibited potent inhibition of tubulin polymerization, binding of\ud colchicine to tubulin, and cancer cell growth. ARDAP derivative 10 inhibited the proliferation of BCR/ABL-expressing KU812\ud and LAMA84 cells from chronic myeloid leukemia (CML) patients in blast crisis and of hematopoietic cells ectopically\ud expressing the imatinib mesylate (IM)-sensitive KBM5-WT or its IM-resistant KBM5-T315I mutation. Compound 10 minimally\ud affected the proliferation of normal blood cells, indicating that it may be a promising agent to overcome broad tyrosine kinase\ud inhibitor resistance in relapsed/refractory CML patients. Compound 10 significantly decreased CML proliferation by inducing\ud G2/M phase arrest and apoptosis via a mitochondria-dependent pathway. ARDAP 10 augmented the cytotoxic effects of IM in\ud human CML cells. Compound 10 represents a robust lead compound to develop tubulin inhibitors with potential as novel treatments for CML.

A cDNA of 312 bp, similar to polygalacturonase-inhibiting proteins (PGIPs), was isolated by cDNA-amplified fragment length polymorphism (cDNA-AFLP) from pea roots infected with the cyst nematode Heterodera goettingiana. The deduced amino acid sequence obtained from the complete Pspgip1 coding sequence was very similar to PGIPs described from several other plant species, and was identical in both MG103738 and Progress 9 genotypes, resistant and susceptible to H. goettingiana, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) expression analysis revealed the differential regulation of the Pspgip1 gene in the two genotypes in response to wounding and nematode challenge. Mechanical wounding induced Pspgip1 expression in MG103738 within 8 h, but this response was delayed in Progress 9. In contrast, the response to nematode infection was more complex. The transcription of Pspgip1 was triggered rapidly in both genotypes, but the expression level returned to levels observed in uninfected plants more quickly in susceptible than in resistant roots. In addition, in situ hybridization showed that Pspgip1 was expressed in the cortical cells damaged as a result of nematode invasion in both genotypes. However, it was specifically localized in the cells bordering the nematode-induced syncytia in resistant roots. This suggests a role for this gene in counteracting nematode establishment inside the root.

Systemic infections are often associated with neurodegenerative processes in many diseases of the central nervous system (CNS), including Parkinson’s disease. Toll-like receptor (TLR)4 and CD14 act as receptors for lipopolysaccharide (LPS) released by gram-negative bacteria. In this contest, CD14 functions as the main LPS ligand and TLR4 transmits the LPS signal into the cell. In this paper, we investigated the expression of TLR4 and CD14, in different anatomical areas of the CNS, in an experimental model of Parkinson’s-like disease, represented by MPTP-treated mouse. In particular, we analyzed the gene transcripts and proteins expression of CD14 and TLR4, in the substantia nigra and caudate-putamen nuclei of these animals. Results demonstrated an augmented expression of both CD14 and TLR4 in the substantia nigra of mice treated with MPTP in comparison to untreated animals, suggesting that the endotoxin receptors are over expressed in different manner in specific areas of the CNS during Parkinson’s-like disease.

Tryptase(+) mast cells (MCs), abundant in the invasive front of tumours, contribute to tissue remodelling. Indeed, protease-activated receptor- 2 (PAR-2) activation by MC-tryptase is considered an oncogenic event in colorectal cancer (CRC). Recently, we have suggested NHERF1 as a potential new marker in CRC. In this study, we aimed to determine the distribution of tryptase(+) MCs and PAR-2 and to examine the relationship between PAR-2 and NHERF1, investigating their reputed usefulness as tumour markers. We studied a cohort of 115 CRC specimens including primary cancer (C) and adjacent normal mucosa (NM) by immunohistochemical double staining, analyzing the protein expression of MC-tryptase, PAR-2 and cytoplasmic NHERF1. MC density was higher in NM than in C. Tumours with high TNM stage and poor grade showed the highest MC density. A higher PAR-2 immunoreactivity characterized tumours most infiltrated by MCs compared with samples with low MC density. Furthermore, PAR-2 overexpression was associated with advanced TNM stage, poor grade and lymphovascular invasion (LVI). A positive correlation existed between tryptase(+) MC density and PAR-2 expression. Cytoplasmic NHERF1 was higher in C than in NM and overexpressing tumours resulted associated with nodal and distant metastases, poor grade and LVI. PAR-2 correlated with cytoplasmic NHERF1 and the PAR-2(+)/cytoplasmic NHERF1(+) expression immunophenotype identified tumours associated with unfavourable prognosis and aggressive clinical parameters. Our data indicate that the high density of tryptase(+) MCs at invasive margins of tumours was associated with advanced stages of CRC and was strongly correlated with PAR-2 expression.

NHERF1 (Na+/H+ exchanger regulatory factor 1) is expressed in the luminal membrane of many epithelia, and associated with proteins involved in tumor progression. Alterations of NHERF1 expression in different sites of metastatic colorectal cancer (mCRC) suggest a dynamic role of this protein in colon carcinogenesis. We focused on the observation of the altered expression of NHERF1 from non-neoplastic tissues to metastatic sites by immunohistochemistry. Moreover, we studied, by immunofluorescence, the colocalization between NHERF1 and the epidermal growth factor receptor (EGFR), whose overexpression is implicated in CRC progression. NHERF1 showed a different localization and expression in the examined sites. The distant non-neoplastic tissues showed NHERF1 mostly expressed at the apical membrane, while in surrounding non-neoplastic tissue decreased the apical membrane and increased cytoplasmic immunoreactivity. In adenomas a shift from apical membrane to cytoplasmic localization and nuclear expression were observed. Cytoplasmic staining in the tumor, and metastatic sites was stronger than surrounding non-neoplastic tissue. Furthermore, nuclear NHERF1 expression was noted in 80% of all samples and surprisingly, it appeared already in adenoma lesions, suggesting that NHERF1 represents an early marker of pre-morphological triggering of colorectal carcinogenesis. Then, in few tumors a positive direct correlation between membrane NHERF1 and EGFR expression was evidenced by their colocalization. Nuclear NHERF1 expression, present in the early stages of carcinogenesis and related with poor prognosis, may contribute to the onset of malignant phenotype. Specifically, we hypothesize the direct involvement of nuclear NHERF1 in both carcinogenesis and progression and its role as a potential colorectal cancer marker.

Parkinson’s disease (PD) is a common neurodegenerative disease characterised by a slow and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Despite intensive research, the cause of neuronal loss in PD is poorly understood. Inflammatory mechanisms have been implicated in the pathophysiology of PD. In this study, conducted on an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we investigated the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and their receptors (IL-1RI, TNF-αRI, IL-6Rα) at the SN and caudate-putamen (CP) levels. In MPTP-treated animals we observed a significant increase in IL-1β, TNF-α and IL-6 mRNA expression levels both in the SN and CP in comparison with untreated mice. In addition, both mRNA and protein levels of IL-1RI, TNF-αRI and IL-6Rα were significantly enhanced in the SN of MPTP-treated mice in comparison to controls, whereas no significant differences were observed in the CP between treated and untreated mice. Overall, these results indicate a role of both pro-inflammatory cytokines and their receptors in the pathogenesis of PD.

Background Traditional determinants proven to be of prognostic importance in breast cancer include the TNM staging, histological grade, proliferative activity, hormone receptor status and HER2 overexpression. One of the limitations of the histological grading scheme is that a high percentage of breast cancers are still classified as grade 2, a category with ambiguous clinical significance. The aim of this study was to best characterize tumors scored as grade 2. Methods We investigated traditional prognostic factors and a panel of tumor markers not used in routine diagnosis, such as NHERF1, VEGFR1, HIF-1α and TWIST1, in 187 primary invasive breast cancers by immunohistochemistry, stratifying patients into good and poor prognostic groups by the Nottingham Prognostic Index. Results Grade 2 subgroup analysis showed that the PVI (p = 0.023) and the loss of membranous NHERF1 (p = 0.028) were adverse prognostic factors. Relevantly, 72% of grade 2 tumors were associated to PVI+/membranous NHERF1- expression phenotype, characterizing an adverse prognosis (p = 0.000). Multivariate logistic regression analysis in the whole series revealed poor prognosis correlated with PVI and MIB1 (p = 0.000 and p = 0.001, respectively). Furthermore, in the whole series of breast cancers we found cytoplasmic NHERF1 expression positively correlated to VEGFR1 (r = 0.382, p = 0.000), and in VEGFR1-overexpressing tumors the oncogenic receptor co-localized with NHERF1 at cytoplasmic level. Conclusions The PVI+/membranous NHERF1- expression phenotype identifies a category of grade 2 tumors with the worst prognosis, including patient subgroup with a family history of breast cancer. These observations support the idea of the PVI+/membranous NHERF1- expression immunophenotype as a useful marker, which could improve the accuracy of predicting clinical outcome in grade 2 tumors.