Biology subjects

Ghosh, M. K.

Publications and source records attributed to Ghosh, M. K..

8 recordsLinked to original sources

bioRxiv2025

CK2α phosphorylates CHIP for oncogenesis: A novel post-translational switch in cancer

The post-translational regulation of tumor suppressors by oncogenic kinases remains a critical yet underexplored determinant of proteostasis reprogramming in cancer. Here, we identified the E3 ubiquitin ligase CHIP (C-terminus of Hsc70-Interacting Protein) as a direct and functionally relevant substrate of the serine/threonine kinase CK2, which is frequently overexpressed in solid tumors. Using LC/MS analysis, in silico kinase prediction, and molecular interaction mapping, we demonstrated that CK2 phosphorylates CHIP at serine 19 (represented as S19), a conserved residue within its TPR domain, thereby promoting ubiquitination and subsequent proteasomal degradation of CHIP. This phosphorylation-dependent destabilization of CHIP impairs its ability to target oncogenic substrates, such as AKT, resulting in sustained AKT phosphorylation and activation, required for oncogenesis. Clinically, we observed a robust inverse correlation between CK2 and CHIP expressions across colorectal and breast cancer patient datasets, which was validated by immunofluorescence (IF) analyses in tumor samples. Multiple functional assays revealed that CK2 suppression, either by genetic ablation or pharmacological inhibition by TBCA, restores CHIP stability, reactivates apoptotic signalling cascades, and attenuates tumor cell proliferation, migration, and 3D spheroid integrity. Moreover, CK2 blockade in syngeneic murine models diminishes primary tumor burden and metastatic dissemination, concomitant with increased CHIP accumulation and reduced AKT signalling. Mechanistically, a phosphorylation-resistant mutant of CHIP at S19 (CHIP-S19A), is refractory to CK2- mediated degradation and preserves anti-tumor functions, delineating a phosphorylation-dependent proteolytic switch as a central node in the CK2-CHIP-AKT regulatory axis. These findings establish a novel paradigm wherein an oncogenic kinase hijacks protein quality control pathway to suppress tumor suppressive activity and facilitate malignancy. Our study positions CK2 as a druggable modulator of CHIP turnover, offering a translational framework for restoring proteostatic checkpoints and constraining oncogenic signalling in aggressive cancers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/682604v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@8fad58org.highwire.dtl.DTLVardef@bd73dforg.highwire.dtl.DTLVardef@2746dorg.highwire.dtl.DTLVardef@129b6ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

PLGA-based dual-loaded nanoformulation of DIM and TMZ - An advanced clinical strategy for brain cancer treatment in a combinatorial approach

Glioblastoma multiforme (GBM) is a highly aggressive form of primary brain tumor in adults, which unfortunately has an abysmal prognosis and poor survival rates. Even though several FDA-approved multimodal treatments for targeting GBM are available, the effectiveness in most patients are not satisfactory. The reason behind this poor success rate is mainly attributed to insufficient drug distribution to the tumor site across the blood-brain barrier (BBB) and induction of resistance for single-drug based therapies. Chemotherapy with Temozolomide (TMZ) having a median overall survival of around 12-15 months, envisages the urgent necessity for more effective treatment strategies. Based upon these facts, in this study, we have developed a novel approach for repurposing TMZ along with inhibition of EGFR, which overexpressed in GBM, to achieve our goal. PLGA-based nanoencapsulation of both TMZ and 3,3-diindoyl methane (DIM), an EGFR inhibitor, in a combinatorial approach enhances the delivery of them together. Their synergistic mode of actions, significantly enhances the cytotoxic effect of TMZ in vitro and in vivo. Moreover, the dual-loaded nanoformulation works more efficiently than their individually packed nanoparticles on DNA damage and apoptosis, resulting in a several-fold reduction in tumor burden, systemic drug toxicity, and increased survival. These findings suggest the preclinical potential of this new treatment strategy.

cancer biology↗

bioRxiv2023

DDX5 (p68) orchestrates β-catenin, RelA and SP1 mediated MGMT gene expression: Implication in TMZ chemoresistance

DDX5 (p68) upregulation has been linked with various cancers of different origins, especially Colon Adenocarcinomas. Similarly, across cancers, MGMT has been identified as the major contributor of chemoresistance against DNA alkylating agents like Temozolomide (TMZ). TMZ is an emerging potent chemotherapeutic agent across cancers under the arena of drug repurposing. Recent studies have established that patients with open MGMT promoters are prone to be innately resistant or acquire resistance against TMZ compared to its closed conformation. However, not much is known about the transcriptional regulation of MGMT gene in the context of colon cancer. This necessitates studying MGMT gene regulation, which directly impacts the cellular potential to develop chemoresistance against alkylating agents. Our study aims to uncover an unidentified mechanism of DDX5-mediated MGMT gene regulation. Experimentally, we found that both mRNA and protein expression levels of MGMT were elevated in response to p68 overexpression in multiple human colon cancer cell lines and vice-versa. Since p68 cannot directly interact with the MGMT promoter, transcription factors viz., {beta}-catenin, RelA (p65) and SP1 were also studied as reported contributors. Through co-immunoprecipitation and GST-pull-down studies, p68 was established as an interacting partner of SP1 in addition to {beta}-catenin and NF-{kappa}B (p50-p65). Mechanistically, luciferase reporter and chromatin-immunoprecipitation assays demonstrated that p68 interacts with the MGMT promoter via TCF4-LEF, RELA and SP1 sites to enhance its transcription. To the best of our knowledge, this is the first report of p68 as a transcriptional co-activator of MGMT promoter and our study identifies p68 as a novel and master regulator of MGMT gene expression.

cancer biology↗

bioRxiv2023

A novel role of USP7 in imparting partial EMT state in colorectal cancer through the DDX3X-β-catenin axis

Epithelial mesenchymal transition (EMT) is a fundamental and highly regulated process that is normally observed during embryonic development and tissue repair but is deregulated during advanced cancer. Classically, through the process of EMT, cancer cells gradually transition from a predominantly epithelial phenotype to a more invasive mesenchymal phenotype. Increasing studies have, however, brought into light the existence of unique intermediary states in EMT, often referred to as partial EMT states. Through our studies we have found the deubiquitinase USP7 to be strongly associated with the development of such a partial EMT state in colon cancer cells, characterized by the acquisition of mesenchymal characteristics but without the reduction in epithelial markers. We found USP7 to be overexpressed in colon adenocarcinomas and to be closely associated with advancing tumor stage. We found that functional inhibition or knockdown of USP7 is associated with a marked reduction in mesenchymal markers and in overall migration potential of cancer cells. Starting off with a proteomics-based approach we were able to identify and later on verify the DEAD box RNA helicase DDX3X to be an interacting partner of USP7. We then went on to show that USP7, through the stabilization of DDX3X, augments Wnt/{beta}-catenin signaling, which has previously been shown to be greatly associated with colorectal cancer cell invasiveness. Our results strongly suggest a positive role of USP7 in the development of a partial mesenchymal phenotype in colorectal cancer.

cancer biology↗

bioRxiv2023

E3 Ubiquitin Ligase CHIP Drives Monoubiquitination-mediated Nuclear Import of Tumor Suppressor PTEN

Nuclear localization of Phosphatase and Tensin Homolog Deleted on Chromosome TEN (PTEN) is crucial for its tumor suppressive activities. Monoubiquitination has been established as a principal mechanism driving nuclear translocation of PTEN. In this study, we describe a novel mechanism wherein Carboxy terminus of Hsc70 Interacting Protein (CHIP) mediates monoubiquitination of PTEN, leading to the latters nuclear import. Analysis of subcellular fractions of multiple cell lines revealed a rise in both nuclear and total cellular PTEN levels under monoubiquitination-promoting conditions, an effect that was abrogated when CHIP expression was silenced. We established a time-point kinetics of CHIP-mediated nuclear translocation of PTEN using immunocytochemistry. We identified a role of Karyopherin 1 (KPNA1) in facilitating nuclear transport of monoubiquitinated PTEN. We further established a direct interaction between CHIP and PTEN inside the nuclear compartment, where CHIP could participate in either polyubiquitination or monoubiquitination of the latter. CHIP was found to monoubiquitinate PTEN on K13 and K289 residues. Finally, we showed that CHIP-mediated monoubiquitination of PTEN resulted in increased apoptosis, decreased cell viability and proliferation. This enhancement of PTENs tumor suppressive abilities by CHIP involved modulation of transcriptional activities of PTEN. To the best of our knowledge, this is the first report elucidating CHIPs non-canonical roles on PTEN, which is also established here as a nuclear interacting partner of CHIP.

cancer biology↗

bioRxiv2021

Identification and validation of E3 ubiquitin ligase XIAP as a novel substrate of deubiquitinase USP7 (HAUSP) - Implication towards oncogenesis

The induction of apoptosis upon USP7 (HAUSP) inhibition is established in cancers that contain a wild-type p53 (p53Wt) through the USP7-Mdm2-p53 axis, but no clear explanation has yet been reported for the same to occur in cancers containing mutant 53 (p53Mut) or even p53 null (p53Null) systems. Instead of this USP7-Mdm2-p53 axis USP7 also works through an alternative new pathway identified in this study. Here in this study, we observed that the magnitude of apoptosis induction in response to USP7 inhibition was remarkably similar between cancer cells showing p53Null or p53Mut and those with p53Wt. Through a proteomics-based approach, we were able to identify XIAP as a novel interacting partner for USP7. XIAP is a potent and well-characterized member of the inhibitor of apoptosis proteins (IAPs), which function through caspase inhibition. We successfully identified USP7 as a positive regulator of XIAP at post-translational but not at its transcriptional level. Using molecular modelling coupled with domain deletion studies, we show that the first three Ubl domains in association with the catalytic domain of USP7 interact with the BIR2 and the linker region between BIR2 and BIR3 domains of XIAP. Modulation of expression and catalytic activity of USP7 in multiple type of cancer cell lines showed that USP7 stabilizes XIAP through its deubiquitinase activity. We have also observed that USP7 sensitizes cells against chemotherapeutic drugs through stabilization of XIAP. Thus, USP7 promotes tumorigenesis in multiple cancers, via stabilization of XIAP that results in apoptosis inhibition in caspase dependent pathway. Moreover, we observed that combinatorial inhibition of USP7 and XIAP can induce cellular apoptosis in a higher magnitude than their individual inhibition. Additionally, our results indicates that nanoformulated P5091 and P22077 showed higher potency for killing C6 cells in comparison to normal drugs. To the best of our knowledge, this is the first report on identification and validation of XIAP, a crucial E3 ubiquitin ligase, as a novel substrate of the deubiquitinase USP7 and they together involve in empowerment of the tumorigenic potential of cancer cells.

cancer biology↗

bioRxiv2021

Wnt/β-catenin signaling and p68 conjointly regulate CHIP in colorectal cancer

The differential expression pattern of Carboxy terminus of Hsc70 Interacting Protein (CHIP, alias STIP1 Homology and U-box Containing Protein 1 or STUB1) in cancers is associated with ubiquitination mediated degradation of its client proteins. Emerging evidences suggest its abundant expression of CHIP in colorectal cancer compared to normal tissues, but the mechanistic detail of this augmented expression pattern is unclear. The signature driver of canonical Wnt pathway, {beta}-catenin, and its co-activator RNA helicase p68, are also overexpressed in colorectal cancer. In this study, we describe a novel mechanism of Wnt/{beta}-catenin and p68 mediated transcriptional activation of CHIP gene leading to enhanced proliferation of colorectal cancer cells. Wnt3A treatment and pharmacological activation of canonical Wnt signaling pathway resulted in increased nuclear translocation of {beta}-catenin and elevated expression of CHIP. Likewise, overexpression and knockdown of {beta}-catenin and p68 upregulated and downregulated CHIP expression, respectively, at both mRNA and protein levels. After cloning CHIP promoter, the increased and decreased promoter activities of CHIP induced by overexpression and knockdown of either {beta}-catenin or p68 further confirmed transcriptional regulation of CHIP gene by Wnt/{beta}-catenin signaling cascade. p68 along with {beta}-catenin were found to occupy Transcription Factor 4 (TCF4) binding sites on endogenous CHIP promoter and regulate its transcription. Finally, enhanced cellular propagation and migration of colorectal cancer cells induced by Wnt/{beta}-catenin-p68-CHIP axis established the significance of this pathway in oncogenesis. To the best of our knowledge, this is the first report elucidating the mechanistic details of transcriptional regulation of CHIP (STUB1) gene expression.

cancer biology↗

bioRxiv2020

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

Iron-sulfur (Fe-S) clusters have a key role in many biochemical processes and are essential for most life forms. Despite recent mechanistic advances in understanding the Fe-S cluster biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share [~]60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreichs ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex reveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been proposed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates for these lost interactions and facilitates a subunit rearrangement to activate the complex. Here, we used a structure and evolution-guided approach to identify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalytic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5-phosphate intermediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system and supporting the architectural switch model. Overall, these studies indicate a weakening of the homodimeric interface was a key development during the evolution of the eukaryotic system and provide new insights into the role of FXN.

biochemistry↗