Elena Lastraioli

Background: KRAS mutations are common oncogenic drivers in metastatic lung cancer (mLC) and may have prognostic and therapeutic implications. Circulating tumor DNA (ctDNA) analysis offers a minimally invasive approach for detecting tumor-specific alterations and monitoring tumor dynamics. This pilot study investigated the concordance between tissue and plasma KRAS mutational status and evaluated its clinical significance. Methods: Forty patients with mLC (25 males, 15 females; mean age 70.8 years) were included. KRAS mutations were analyzed in formalin-fixed paraffin-embedded tumor tissue using next-generation sequencing and in baseline plasma ctDNA using the Biocartis Idylla™ system. Concordance was assessed using Cohen’s kappa statistics. Associations with clinicopathological characteristics and survival outcomes (overall survival (OS), progression-free survival (PFS), and time to progression (TTP)) were examined. Longitudinal ctDNA monitoring was performed in a subset of patients. Results: KRAS mutations were detected in 14/36 (38.89%) of tumor tissues and 12/40 (30.00%) of plasma samples, predominantly involving codon 12, with G12C as the most frequent variant. Tissue–plasma concordance was assessed in the 36 patients with matched samples and was substantial (κ = 0.64), while mutation subtype showed almost perfect agreement among concordant cases (percentage of agreement: 88.9%, κ = 0.80, p = 0.002, 95% CI: 0.4400–1.0000). No significant associations were observed with clinicopathological variables. Longitudinal analysis revealed dynamic ctDNA changes, with molecular progression occasionally preceding clinical progression. Patients with KRAS-mutated ctDNA had shorter PFS and OS compared to those with wild-type ctDNA (median PFS: 134 vs. 146 days; median OS: 163 vs. 193 days). Conclusions: Plasma ctDNA detection of KRAS mutations identifies a poor-prognosis subgroup of mLC patients and provides dynamic insights into tumor evolution. Serial ctDNA monitoring may anticipate disease progression and support real-time assessment of treatment response, highlighting its potential role in translational research and precision oncology.

Mounting evidence underline the relevance of macromolecular complexes in cancer. Integrins frequently recruit ion channels and transporters within complexes which behave as signaling hubs. A complex composed by β1 integrin, hERG1 K+ channel, the neonatal form of the Na+ channel NaV 1.5 (nNaV1.5) and the Na+/H+ antiporter NHE1 (NHE1/hERG1/β1/nNaV1.5 complex) has been recently described to be expressed and regulate relevant cancer related behaviors in Breast Cancer (BCa) cells. We analyzed the expression and impact on outcome of the genes encoding the four proteins forming the NHE1/hERG1/β1/nNaV1.5 complex (SLC9A1, KCNH2, ITGB1 and SCN5A) in public datasets. The corresponding proteins were also evaluated by immunohistochemistry and their expression was correlated with clinic-pathological and molecular characteristics and patients’ survival. The expression of KCNH2 and SCN5A was significantly correlated in primary BCa as occurs in the heart, although with a broader distribution, forming a functional network which also included ITGB1 and SLC9A1. The co-expression proteins emerged from the immunohistochemistry analysis. Interestingly, hERG1, nNav1.5 and the hERG1/β1 integrin complex associated with several clinical features, including molecular subtype and hormone receptor status. Moreover, hERG1 and the combination of hERG1 and nNav1.5 had impact on prognosis, contributing to identifying a group of patients with worse prognosis. hERG1 and nNav1.5 channels along with β1 integrins and the NHE1 antiporter are co-expressed in BCa both at gene and protein levels, assembling into a macromolecular complex. The NHE1/hERG1/β1/nNaV1.5 complex can be considered a novel biomarker and potential target for therapy for BCa patients.

Reciprocal signaling between the Tumor Microenvironment (TME) and cancer cells regulates abnormal proliferation, migration and pro-metastatic behavior. Major player in such interaction is integrin-mediated cell adhesion to the Extracellular Matrix (ECM). Integrin receptors organize signaling hubs constituted by multiprotein membrane complexes often comprising ion channels and transporters. We studied whether and how integrin-centered multiprotein complexes control cell behavior in Breast Cancer (BCa) cell populations with different molecular characteristics. BCa cells were cultured onto the ECM protein fibronectin (FN), to trigger β1 integrin activation. Through biochemical, immunofluorescence and electrophysiological experiments we provide evidence of a novel signaling pathway that involves a β1 integrin-centered plasma membrane complex formed by different transport proteins: the hERG1 K ⁺ channel, the neonatal form of the Na ⁺ channel Na _V 1.5 (nNa _V 1.5) and the Na ⁺ /H ⁺ antiporter NHE1. The NHE1/hERG1/β1/nNa _V 1.5 complex was found on the plasma membrane of BCa cells, and particularly of Triple Negative Breast Cancer (TNBCa). When engaged by cell adhesion to FN, such membrane complex recruited the cytoskeletal actin-binding protein a-actinin1 and stimulated NHE1-mediated cytoplasmic alkalinization. Thus, the multiprotein complex activation affected TNBCa migration and invasiveness by stimulating f-actin organization directly (through α-actinin1) and indirectly (by intracellular alkalinization). The contribution of both hERG1 and nNa _V 1.5 was essential, as the adhesion-dependent signaling pathway and its functional consequences were inhibited by blocking either channel with, respectively, E4031 and TTX, or by applying RNA silencing procedures. The contribution of hERG1 to the structural integrity of the membrane complex appeared to be critical, as the adhesion-dependent signals were hampered by harnessing the hERG1/β1 integrin complex with a single chain bispecific antibody (scDb-hERG1-β1) which disrupts the macromolecular complex without blocking the K ⁺ current, as well as by E4031, which impairs the complex formation by blocking the channel in the open state. In conclusion, we revealed that integrin-centered macromolecular complexes in BCa cells recruit a battery of ion transport proteins that cooperate in modulating different aspects of the downstream signals that lead to malignant behavior. This complex could be targeted to develop novel therapeutic strategies for one of the most difficult-to-treat cancers, i.e. TNBCa.

The hERG1 potassium channel is aberrantly over expressed in tumors and regulates the cancer cell response to integrin-dependent adhesion. We unravel a novel signaling pathway by which integrin engagement by the ECM protein fibronectin promotes hERG1 translocation to the plasma membrane and its association with β1 integrins, by activating girdin-dependent Gαi3 proteins and protein kinase B (Akt). By sequestering hERG1, β1 integrins make it avoid Rab5-mediated endocytosis, where unbound channels are degraded. The cycle of hERG1 expression determines the resting potential (V_rest) oscillations and drives the cortical f-actin dynamics and thus cell motility. To interpret the slow biphasic kinetics of hERG1/β1 integrin interplay, we developed a mathematical model based on a generic balanced inactivation–like module. Integrin-mediated cell adhesion triggers two contrary responses: a rapid stimulation of hERG1/β1 complex formation, followed by a slow inhibition which restores the initial condition. The protracted hERG1/β1 integrin cycle determines the slow time course and cyclic behavior of cell migration in cancer cells.

Despite improvements in treatment, lung cancer is still a major health problem worldwide. Among lung cancer subtypes, the most frequent is represented by adenocarcinoma (belonging to the Non-Small Cell Lung Cancer class) although the most challenging and harder to treat is represented by Small Cell Lung Cancer, that occurs at lower frequency but has the worst prognosis. For these reasons, the standard of care for these patients is represented by a combination of surgery, radiation therapy and chemotherapy. In this view, searching for novel biomarkers that might help both in diagnosis and therapy is mandatory. In the last 30 years it was demonstrated that different families of ion channels are overexpressed in both lung cancer cell lines and primary tumours. The altered ion channel profile may be advantageous for diagnostic and therapeutic purposes since most of them are localised on the plasma membrane thus their detection is quite easy, as well as their block with specific drugs and antibodies. This review focuses on ion channels (Potassium, Sodium, Calcium, Chloride, Anion and Nicotinic Acetylcholine receptors) in lung cancer (both Non-Small Cell Lung Cancer and Small Cell Lung Cancer) and recapitulate the up-to-date knowledge about their role and clinical relevance for a potential use in the clinical setting, for lung cancer diagnosis and therapy.

In this study we evaluated both~ K- and N-RAS mutations in plasma samples from patients with metastatic colorectal cancer by means of the BEAMing technology, and we assessed their diagnostic performance compared to RAS analyses performed on tissue. The sensitivity of BEAMing in identifying KRAS mutations was of 89.5%, with a fair specificity. The agreement with tissue analysis was moderate. The sensitivity for NRAS was high with a good specificity, and the agreement between tissue analysis and BEAMing was fair. Interestingly, significantly higher mutant allele fraction (MAF) levels were detected in patients with G2 tumors, liver metastases, and in those who did not receive surgery. NRAS MAF level was significantly higher in patients with mucinous adenocarcinoma and for those with lung metastases. A sharp increase in the MAF values was observed in patients who moved towards disease progression. More strikingly, molecular progression always anticipated the radiological one in these patients. These observations pave the way to the possibility of using liquid biopsy to monitor patients during treatment, and to enable oncologists to anticipate interventions compared to radiological analyses. This will allow time to be saved and ensure a better management of metastatic patients in the near future.

MAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of this study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the β1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells. We provide evidence that the scDb we produced binds to the hERG1/β1 complex in cancer cells and tissues, but does not bind to the hERG1 channel in nonpathologic tissues, in particular the heart. The scDb-hERG1-β1 (i) downregulates the formation of the hERG1/β1 complex, (ii) inhibits Akt phosphorylation and HIF-1α expression, and (iii) decreases cell survival, proliferation, and migration in vitro. These effects only occur in cancer cells (either colon, pancreatic, or breast), but not in normal cells. In vivo, the scDb-hERG1-β1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity when injected intravenously up to the dose of 8 mg/kg. The scDb-hERG1-β1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization. Overall, the scDb-hERG1-β1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers that overexpress the hERG1/β1 integrin signaling complex.