Targeting KRAS in pancreatic cancer

Pancreatic cancer has a dismal prognosis due to late detection and lack of efficient therapies.The Kirsten rat sarcoma virus(KRAS)oncogene is mutated in up to 90%of all pancreatic ductal adenocarcinomas(PDACs)and constitutes an attractive target for therapy.However,the most common KRAS mutations in PDAC are G12D(44%),G12V(34%)and G12R(20%)that are not amenable to treatment by KRAS G12C-directed cysteine-reactive KRAS inhibitors such as Sotorasib and Adagrasib that exhibit clinical efficacy in lung cancer.KRAS G12C mutant pancreatic cancer has been treated with Sotorasib but this mutation is detected only in 2%–3%of PDAC.Recently,the KRAS G12D-directed MRTX1133 inhibitor has entered clinical trials and more of such inhibitors are in development.The other KRAS mutations may be targeted indirectly via inhibition of the cognate guanosine exchange factor(GEF)Son of Sevenless 1 that drives KRAS.These agents seem to provide the means to target the most frequent KRAS mutations in PDAC and to improve patient outcomes.

Changes of protein expression during tumorosphere formation of small cell lung cancer circulating tumor cells

Small cell lung cancer(SCLC)is frequently disseminated and has a dismal prognosis with survival times of approximately two years.This cancer responds well to initial chemotherapy but recurs within a short time as a globally chemoresistant tumor.Circulating tumor cells(CTCs)are held responsible for metastasis,the extremely high numbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines.These CTCs are distinguished by the spontaneous formation of large spheroids,termed tumorospheres,in regular tissue culture.These contain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to single cell cultures.Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as single cells or in the form of tumorospheres in Western blot arrays.With the exception of the UHGc5 line,all other CTC lines express EpCAM and lack a complete EpCAM-negative,vimentin-positive epithelial-mesenchymal transition(EMT)phenotype.Upon formation of tumorospheres the expression of EpCAM,that mediates cell-cell adhesion is markedly upregulated.Proteins such as E-Cadherin,p27 KIP1,Progranulin,BXclx,Galectin-3,and Survivin showed variable changes for the distinct CTC cell lines.In conclusion,EpCAM presents the most critical marker for individual SCLC CTCs and the assembly of highly chemoresistant tumorospheres.

Protection of small-cell lung cancer circulating tumor cells by cellular fragmentation

Aim:Small-cell lung cancer(SCLC)disseminates aggressively and may exhibit high chemoresistance and poor survival rates.In this study,we aimed to investigate a new mechanism of drug resistance for SCLC circulating tumor cells(CTCs).Methods:SCLC CTC cell lines(n=4)which shed cellular fragments(MAT),as demonstrated by light and scanning electron microscopy,are compared to permanent SCLC lines.Selected proteins are detected by proteome arrays and the functional impact of MAT is studied using cytotoxicity tests involving cisplatin and Topotecan.Results:The SCLC CTC lines revealed layers of attached cellular fragments with a range of decreasing sizes from intact cells(approximately 12μm)down to small debris(approximately 2μm)which are not detectable in permanent SCLC lines.Intact SCLC CTC clusters represent cores of these fragment-coated spheroids.Proteome profiling of MAT revealed a protein pattern similar to intact cells.Chemosensitivity tests employing SCLC and SCLC CTC lines with chemotherapeutics used in therapy of SCLC demonstrated an inhibitory activity of MAT on the resulting cytotoxicity.Conclusion:Generation of cell-associated debris by SCLC CTCs offers protective effects against cytotoxic drugs,representing a novel mechanism allowing survival of SCLC CTCs in patients.

Role of circulating tumor cell spheroids in drug resistance

Cancer cell spheroids are used for drug screening as these three-dimensional(3D)assemblies recapitulate tumors more realistic than the widely employed 2D in vitro cultures.Limited drug diffusion and gradients of oxygen and nutrients in spheroids represent avascular tumor regions containing quiescent and hypoxic tumor cells with high drug resistance.Circulating tumor cells(CTCs)effect metastatic spread and are present in high numbers in malignant diseases such as small-cell lung cancer(SCLC)and in other cancer patients with high tumor load.CTCs are heterogeneous and only a small fraction of these cells survive in the circulation and cause distal lesions.CTCs may circulate as single cells but small CTC clusters or CTC spheroids have been detected in cancer patients and demonstrated to possess increased metastatic potential.At our lab we have obtained 9 permanent SCLC CTC cell lines(BHGc7,BHGc10,BHGc16,BHGc26,BHGc27,BHGc50,BHGc59,BHGc71,and UHGc5)of distinct patients exhibiting similar characteristics and spontaneous formation of large spheroids,termed tumorospheres.These aggregates were shown to exhibit high drug resistance compared to the corresponding single cell suspensions.The increased metastatic capability of small circulating tumor clusters/spheroids may be explained by their role as putative precursors of tumorospheres eventually trapped in capillaries.Limited drug penetration and the presence of hypoxic/quiescent cells can readily account for the global drug resistance of advanced SCLC which has resulted in clinical failure of a wide range of chemotherapeutics and low survival.Furthermore,we have detected such tumorospheres in non-small-cell lung cancer(NSCLC)patients progressing under EGFR-directed tyrosine kinase inhibitor therapy which had undergone NSCLC-SCLC transformation.

Non-small cell lung cancer-small cell lung cancer transformation as mechanism of resistance to tyrosine kinase inhibitors in lung cancer

Mutated or rearranged driver kinases in non-small cell lung cancer(NSCLC)cells are clinically amenable to treatment with tyrosine kinase inhibitors(TKIs)resulting in prolonged survival and significant benefit compared to cytotoxic chemotherapy.The most frequent genomic alterations are observed for epidermal growth factor receptor and anaplastic lymphoma kinase,which can be blocked by a range of specific TKIs in sequence.In clinics,resistance to TKIs emerges after approximately one year and comprises secondary mutations of the kinases(on-target)or alternative pathways circumventing the original kinase(off-target)alterations.A special feature of NSCLC is the occurrence of histological transformation to small cell lung cancer(SCLC)in up to 14%of cases,which,in general,is accompanied by resistance to the original TKIs.SCLC transformed tumors may be treated with the classical platinum/etoposide regimen but thus far there are no definitive guidelines.Four transformed pleural SCLC lines in our lab indicate the presence of a gradual NSCLC-SCLC shift with overlapping drug sensitivities.In conclusion,the treatment of NSCLC-SCLC transformed cancer cells would need a better chemosensitivity assessment using functional genomics to guide further therapy.

Insights into mechanisms of tumor dissemination from circulating tumor cell lines of small cell lung cancer

Despite the fact that the majority of cancer patients succumb to metastatic disease,most aspects of tumor metastasis are not understood in detail at present.Cell biologic steps of dissemination are difficult to characterize in human tumors and research is in large part confined to cell line and experimental animal studies.Epithelial-mesenchymal transition(EMT),intravasation of malignant cells,dissemination as circulating tumor cells(CTCs)and eventually mesenchymal-epithelial transition(MET)at distal sites are steps believed to be involved in metastasis.Small cell lung cancer(SCLC)is distinguished by early dissemination and excessive numbers of CTCs,which allowed for the ex vivo expansion of six permanent CTC lines taken from relapsed patients.Cells exhibit an epithelial phenotype with partial EMT traits and are chemoresistant due to formation of large tumorospheres.Since cells may have invaded without undergoing EMT,the role of MET is uncertain.These SCLC CTC cell lines seem to represent the metastasis-inducing cancer cells;these are the minute subpopulation of CTCs capable of surviving in the circulation and transitioning to metastases,leading in turn to resistance and failure of therapy.Full characterization of these lines is expected to provide the markers to find the relevant CTCs among the highly heterogeneous population observable in the context of tumor recurrence.