A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis

Abstract

IntroductionWe have demonstrated that transposons derived from ctDNA can be transferred between cancer cells. The present research aimed to investigate the cellular uptake and intracellular trafficking of Multiple Myeloma-zip code (MM-ZC), a cell-specific zip code, in myeloma cell lines. We demonstrated that MM-ZC uptake by myeloma cells was concentration-, time- and cell-type-dependent.MethodsFlow cytometry and confocal microscopy methods were used to identify the level of internalization of the zip codes in MM cells. To screen for the mechanism of internalization, we used multiple inhibitors of endocytosis. These experiments were followed by biotin pulldown and confocal microscopy for validation. Single interference RNA (siRNA) targeting some of the proteins involved in endocytosis was used to validate the role of this pathway in ZC cell internalization.ResultsEndocytosis inhibitors identified that Monensin and Chlorpromazine hydrochloride significantly reduced MM-ZC internalization. These findings suggested that Clathrin-mediated endocytosis and endosomal maturation play a crucial role in the cellular uptake of MM-ZC. Biotin pulldown and confocal microscopic studies revealed the involvement of proteins such as Clathrin, Rab5a, Syntaxin-6, and RCAS1 in facilitating the internalization of MM-ZC. Knockdown of Rab5a and Clathrin proteins reduced cellular uptake of MM-ZC and conclusively demonstrated the involvement of Clathrin-Rab5a pathways in MM-ZC endocytosis. Furthermore, both Rab5a and Clathrin reciprocally affected their association with MM-ZC when we depleted their proteins by siRNAs. Additionally, the loss of Rab5a decreased the Syntaxin-6 association with MMZC but not vice versa. Conversely, MM-ZC treatment enhanced the association between Clathrin and Rab5a.ConclusionOverall, the current study provides valuable insights into the cellular uptake and intracellular trafficking of MM-ZC in myeloma cells. Identifying these mechanisms and molecular players involved in MM-ZC uptake contributes to a better understanding of the delivery and potential applications of cell-specific Zip-Codes in gene delivery and drug targeting in cancer research.

Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells

“…The putative receptor and the consensus ‘antigenic’ sequences in transposons and the mechanism by which ctDNA and the putative receptor interact need to be elucidated. Regarding the intracellular trafficking of ctDNA, our data indicate that the internalization of a ctDNA transposon sequence utilizes clathrin and endosomal maturation pathways similar to those used by aptamers ( 38 , 39 ). However, whether these pathways are universally applicable across various tumor types and for all transposon-associated ctDNA sequences remains to be determined.…”

Unveiling retrotransposon-derived DNA zip code for myeloma cell internalization

“…Our recently published research [ 54 ] sheds light on a novel mechanism by which myeloma cells internalize retrotransposon-derived DNA sequences, offering new insights into the pathogenesis and potential therapeutic targets for multiple myeloma. The study elucidates a previously unrecognized role of retrotransposon-derived DNA elements, in facilitating the internalization of myeloma cells.…”

“…By exploiting endogenous cellular machinery for DNA uptake, myeloma cells may acquire genetic material from the microenvironment, potentially influencing tumor progression, drug resistance, and immune evasion. Moreover, identifying Clathrin-Rab5a-mediated endocytosis ( Figure 1 ) as the mechanism underlying retrotransposon-derived DNA internalization provides [ 54 ] a rationale for exploring targeted interventions to disrupt this process as a therapeutic strategy for multiple myeloma. Small molecule inhibitors or monoclonal antibodies targeting critical components of the endocytic machinery could impede the uptake of exogenous DNA and attenuate myeloma cell proliferation and survival.…”