Clinical and Preclinical Targeting of Oncogenic Pathways in PDAC: Targeted Therapeutic Approaches for the Deadliest Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide. It is commonly diagnosed in advanced stages and therapeutic interventions are typically constrained to systemic chemotherapy, which yields only modest clinical outcomes. In this review, we examine recent developments in targeted therapy tailored to address distinct molecular pathway alteration required for PDAC. Our review delineates the principal signaling pathways and molecular mechanisms implicated in the initiation and progression of PDAC. Subsequently, we provide an overview of prevailing guidelines, ongoing investigations, and prospective research trajectories related to targeted therapeutic interventions, drawing insights from randomized clinical trials and other pertinent studies. This review focus on a comprehensive examination of preclinical and clinical data substantiating the efficacy of these therapeutic modalities, emphasizing the potential of combinatorial regimens and novel therapies to enhance the quality of life for individuals afflicted with PDAC. Lastly, the review delves into the contemporary application and ongoing research endeavors concerning targeted therapy for PDAC. This synthesis serves to bridge the molecular elucidation of PDAC with its clinical implications, the evolution of innovative therapeutic strategies, and the changing landscape of treatment approaches.

Biomarkers of axonal damage to favor early diagnosis in variant transthyretin amyloidosis (A-ATTRv).

Early identification of ATTRv amyloidosis disease onset is still often delayed due to the lack of validated biomarkers of this disease. Light chain neurofilament (NfL) have shown promising results in early diagnosis in this disease, but data is still needed, including with alternative measuring methods. Our aim was to study the levels of NfL measured by ELISA. Furthermore, interstitial matrix metalloproteinase type 1 (MMP-1) serum levels were measured as a potential new biomarker in ATTRv. Serum NfL and MMP-1 were measured using ELISA assays in 90 participants (29 ATTR-V30M patients, 31 asymptomatic V30M-TTR variant carriers and 30 healthy controls). Median NfL levels among ATTRv amyloidosis patients were significantly higher (116 pg/mL vs 0 pg/mL in both comparison groups). The AUC comparing ATTRv amyloidosis patients and asymptomatic carriers was 0.90 and the NfL concentration of 93.55 pg/mL yielded a sensitivity of 79% and a specificity of 87%. NfL levels had a significant positive correlation with NIS values among patients. We found a negative significant correlation between eGFR and NfL levels. Finally, MMP1 levels were not different between groups. Evidence of NfL use for early diagnosis of ATTR-PN amyloidosis is growing. ELISA seems a reliable and available technique for it quantification. Decreased GFR could influence NfL plasma levels.

Challenges in precision medicine in pancreatic cancer: A focus in cancer stem cells and microbiota.

Pancreatic cancer adenocarcinoma (PDAC) is a lethal disease, with the lowest 5-years survival rate of all cancers due to late diagnosis. Despite the advance and success of precision oncology in gastrointestinal cancers, the frequency of molecular-informed therapy decisions in PDAC is currently neglectable. The reasons for this dismal situation are mainly the absence of effective early diagnostic biomarkers and therapy resistance. PDAC cancer stem cells (PDAC-SC), which are regarded as essential for tumor initiation, relapse and drug resistance, are highly dependent on their niche i.e. microanatomical structures of the tumor microenvironment. There is an altered microbiome in PDAC patients embedded within the highly desmoplastic tumor microenvironment, which is known to determine therapeutic responses and affecting survival in PDAC patients. We consider that understanding the communication network that exists between the microbiome and the PDAC-SC niche by co-culture of patient-derived organoids (PDOs) with TME microbiota would recapitulate the complexity of PDAC paving the way towards a precision oncology treatment-response prediction.

Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells.

Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

GDF15 and ACE2 stratify COVID-19 patients according to severity while ACE2 mutations increase infection susceptibility.

Coronavirus disease 19 (COVID-19) is a persistent global pandemic with a very heterogeneous disease presentation ranging from a mild disease to dismal prognosis. Early detection of sensitivity and severity of COVID-19 is essential for the development of new treatments. In the present study, we measured the levels of circulating growth differentiation factor 15 (GDF15) and angiotensin-converting enzyme 2 (ACE2) in plasma of severity-stratified COVID-19 patients and uninfected control patients and characterized the in vitro effects and cohort frequency of ACE2 SNPs. Our results show that while circulating GDF15 and ACE2 stratify COVID-19 patients according to disease severity, ACE2 missense SNPs constitute a risk factor linked to infection susceptibility.

The Role of LSD1 and LSD2 in Cancers of the Gastrointestinal System: An Update.

Epigenetic mechanisms are known to play a key role in cancer progression. Specifically, histone methylation involves reversible post-translational modification of histones that govern chromatin structure remodelling, genomic imprinting, gene expression, DNA damage repair, and meiotic crossover recombination, among other chromatin-based activities. Demethylases are enzymes that catalyse the demethylation of their substrate using a flavin adenine dinucleotide-dependent amine oxidation process. Lysine-specific demethylase 1 (LSD1) and its homolog, lysine-specific demethylase 2 (LSD2), are overexpressed in a variety of human cancer types and, thus, regulate tumour progression. In this review, we focus on the literature from the last 5 years concerning the role of LSD1 and LSD2 in the main gastrointestinal cancers (i.e., gastric cancer, liver cancer, pancreatic cancer, and colorectal cancer).

Organoids: An Emerging Tool to Study Aging Signature across Human Tissues. Modeling Aging with Patient-Derived Organoids.

The biology of aging is focused on the identification of novel pathways that regulate the underlying processes of aging to develop interventions aimed at delaying the onset and progression of chronic diseases to extend lifespan. However, the research on the aging field has been conducted mainly in animal models, yeast, Caenorhabditis elegans, and cell cultures. Thus, it is unclear to what extent this knowledge is transferable to humans since they might not reflect the complexity of aging in people. An organoid culture is an in vitro 3D cell-culture technology that reproduces the physiological and cellular composition of the tissues and/or organs. This technology is being used in the cancer field to predict the response of a patient-derived tumor to a certain drug or treatment serving as patient stratification and drug-guidance approaches. Modeling aging with patient-derived organoids has a tremendous potential as a preclinical model tool to discover new biomarkers of aging, to predict adverse outcomes during aging, and to design personalized approaches for the prevention and treatment of aging-related diseases and geriatric syndromes. This could represent a novel approach to study chronological and/or biological aging, paving the way to personalized interventions targeting the biology of aging.

KRAS phosphorylation regulates cell polarization and tumorigenic properties in colorectal cancer.

Oncogenic mutations of KRAS are found in the most aggressive human tumors, including colorectal cancer. It has been suggested that oncogenic KRAS phosphorylation at Ser181 modulates its activity and favors cell transformation. Using nonphosphorylatable (S181A), phosphomimetic (S181D), and phospho-/dephosphorylatable (S181) oncogenic KRAS mutants, we analyzed the role of this phosphorylation to the maintenance of tumorigenic properties of colorectal cancer cells. Our data show that the presence of phospho-/dephosphorylatable oncogenic KRAS is required for preserving the epithelial organization of colorectal cancer cells in 3D cultures, and for supporting subcutaneous tumor growth in mice. Interestingly, gene expression differed according to the phosphorylation status of KRAS. In DLD-1 cells, CTNNA1 was only expressed in phospho-/dephosphorylatable oncogenic KRAS-expressing cells, correlating with cell polarization. Moreover, lack of oncogenic KRAS phosphorylation leads to changes in expression of genes related to cell invasion, such as SERPINE1, PRSS1,2,3, and NEO1, and expression of phosphomimetic oncogenic KRAS resulted in diminished expression of genes involved in enterocyte differentiation, such as HNF4G. Finally, the analysis, in a public data set of human colorectal cancer, of the gene expression signatures associated with phosphomimetic and nonphosphorylatable oncogenic KRAS suggests that this post-translational modification regulates tumor progression in patients.

Stromal gene expression defines poor-prognosis subtypes in colorectal cancer.

Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profiles have defined subtypes displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-ß signaling. Likewise, we find that all poor-prognosis CRC subtypes share a gene program induced by TGF-ß in tumor stromal cells. Using patient-derived tumor organoids and xenografts, we show that the use of TGF-ß signaling inhibitors to block the cross-talk between cancer cells and the microenvironment halts disease progression.

Ribonucleoprotein HNRNPA2B1 interacts with and regulates oncogenic KRAS in pancreatic ductal adenocarcinoma cells.

BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDAC) involves activation of c-Ki-ras2 Kirsten rat sarcoma oncogene homolog (KRAS) signaling, but little is known about the roles of proteins that regulate the activity of oncogenic KRAS. We investigated the activities of proteins that interact with KRAS in PDAC cells. METHODS: We used mass spectrometry to demonstrate that heterogeneous nuclear ribonucleoproteins (HNRNP) A2 and B1 (encoded by the gene HNRNPA2B1) interact with KRAS G12V. We used co-immunoprecipitation analyses to study interactions between HNRNPA2B1 and KRAS in KRAS-dependent and KRAS-independent PDAC cell lines. We knocked down HNRNPA2B1 using small hairpin RNAs and measured viability, anchorage-independent proliferation, and growth of xenograft tumors in mice. We studied KRAS phosphorylation using the Phos-tag system. RESULTS: We found that interactions between HRNPA2B1 and KRAS correlated with KRAS-dependency of some human PDAC cell lines. Knock down of HNRNPA2B1 significantly reduced viability, anchorage-independent proliferation, and formation of xenograft tumors by KRAS-dependent PDAC cells. HNRNPA2B1 knock down also increased apoptosis of KRAS-dependent PDAC cells, inactivated c-akt murine thymoma oncogene homolog 1 signaling via mammalian target of rapamycin, and reduced interaction between KRAS and phosphatidylinositide 3-kinase. Interaction between HNRNPA2B1 and KRAS required KRAS phosphorylation at serine 181. CONCLUSIONS: In KRAS-dependent PDAC cell lines, HNRNPA2B1 interacts with and regulates the activity of KRAS G12V and G12D. HNRNPA2B1 is required for KRAS activation of c-akt murine thymoma oncogene homolog 1-mammalian target of rapamycin signaling, interaction with phosphatidylinositide 3-kinase, and PDAC cell survival and tumor formation in mice. HNRNPA2B1 might be a target for treatment of pancreatic cancer.

Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth.

KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors.

Oncogenic K-ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status.

Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

CaM interaction and Ser181 phosphorylation as new K-Ras signaling modulators.

The small G-protein Ras was the first oncogene to be identified and has a very important contribution to human cancer development (20-23% prevalence). K-RasB, one of the members of the Ras family, is the one that is most mutated and plays a prominent role in pancreatic, colon and lung cancer development. Ras proteins are membrane bound GTPases that cycle between inactive, GDP-bound and active, GTP-bound, states. Most of the research into K-RasB activity regulation has focused on the analysis of how GTP-exchange factors (GEFs) and GTPase activating proteins (GAPs) are regulated by external and internal signals. In contrast, oncogenic K-RasB has a very low GTPase activity and furthermore is not deactivated by GAPs. Consequently, the consensus was that activity of oncogenic K-RasB was not modulated. In this extra view we recapitulate some recent data showing that calmodulin binding to K-RasB inhibits phosphorylation of K-RasB at Ser181, near to the membrane anchoring domain, modulating signaling of both non-oncogenic and oncogenic K-RasB. This may be relevant to normal cell physiology, but also opens new therapeutic perspectives for the inhibition of oncogenic K-RasB signaling in tumors.