Colorectal Cancer Immunotherapy: State of the Art and Future Directions.

Cancer immunotherapy has become an indispensable mode of treatment for a multitude of solid tumor cancers. Colorectal cancer (CRC) has been one of the many cancer types to benefit from immunotherapy, especially in advanced disease where standard treatment fails to prevent recurrence or results in poor survival. The efficacy of immunotherapy in CRC has not been without challenge, as early clinical trials observed dismal responses in unselected CRC patients treated with checkpoint inhibitors. Many studies and clinical trials have since refined immunotherapies available for CRC, solidifying immunotherapy as a powerful asset for CRC treatment. This review article examines CRC immunotherapies, from their foundation, through emerging avenues for improvement, to future directions.

HPIP and RUFY3 are noncanonical guanine nucleotide exchange factors of Rab5 to regulate endocytosis-coupled focal adhesion turnover.

While the role of endocytosis in focal adhesion turnover-coupled cell migration has been established in addition to its conventional role in cellular functions, the molecular regulators and precise molecular mechanisms that underlie this process remain largely unknown. In this study, we report that proto-oncoprotein hematopoietic PBX-interacting protein (HPIP) localizes to focal adhesions as well as endosomal compartments along with RUN FYVE domain-containing protein 3 (RUFY3) and Rab5, an early endosomal protein. HPIP contains two coiled-coil domains (CC1 and CC2) that are necessary for its association with Rab5 and RUFY3 as CC domain double mutant, that is, mtHPIPΔCC1-2 failed to support it. Furthermore, we show that HPIP and RUFY3 activate Rab5 by serving as noncanonical guanine nucleotide exchange factors of Rab5. In support of this, either deletion of coiled-coil domains or silencing of HPIP or RUFY3 impairs Rab5 activation and Rab5-dependent cell migration. Mechanistic studies further revealed that loss of HPIP or RUFY3 expression severely impairs Rab5-mediated focal adhesion disassembly, FAK activation, fibronectin-associated-ß1 integrin trafficking, and thus cell migration. Together, this study underscores the importance of HPIP and RUFY3 as noncanonical guanine nucleotide exchange factors of Rab5 and in integrin trafficking and focal adhesion turnover, which implicates in cell migration.

A reciprocal feedback loop between HIF-1α and HPIP controls phenotypic plasticity in breast cancer cells.

While phenotypic plasticity is a critical factor contributing to tumor heterogeneity, molecular mechanisms underlying this process are largely unknown. Here we report that breast cancer cells display phenotypic diversity in response to hypoxia or normoxia microenvironments by operating a reciprocal positive feedback regulation of HPIP and HIF-1α. We show that under hypoxia, HIF-1α induces HPIP expression that establishes cell survival, and also promotes cell migration/invasion, EMT and metastatic phenotypes in breast cancer cells. Mechanistic studies revealed that HPIP interacts with SRP14, a component of signal recognition particle, and stimulates MMP9 synthesis under hypoxic stress. Whereas, in normoxia, HPIP stabilizes HIF-1α, causing the Warburg effect to support cell growth. Concurrently, mathematical modelling corroborates this reciprocal feedback loop in enabling cell-state transitions in cancer cells. Clinical data indicate that elevated levels of HPIP and HIF-1α correlate with unfavorable prognosis and shorter survival rates in breast cancer subjects. Together, this data shows a reciprocal positive feedback loop between HPIP and HIF-1α that was unknown hitherto. It unveils how the tumor microenvironment influences phenotypic plasticity that has an impact on tumor growth and metastasis and, further signifies considering this pathway as a potential therapeutic target in breast cancer.

Hematopoietic PBX-interacting protein is a novel regulator of mammary epithelial cell differentiation.

Hematopoietic PBX-interacting protein (HPIP, also known as PBXIP1) is an estrogen receptor (ER) interacting protein that regulates estrogen-mediated breast cancer cell proliferation and tumorigenesis. However, its functional significance in the context of mammary gland development is unexplored. Here, we report that HPIP is required for prolactin (PRL)-induced lactogenic differentiation in vitro. Molecular analysis of HPIP expression in mice revealed its induced expression at pregnancy and lactation stages of mammary gland. Moreover, PRL is a lactogenic hormone that controls pregnancy as well as lactation and induces Hpip/Pbxip1 expression in a signal transducer and activator of transcription 5a-dependent manner. Using mammary epithelial and lactogenic-competent cell lines, we further show that HPIP plays a regulatory role in PRL-mediated mammary epithelial cell differentiation, which is measured by acini formation, ß-casein synthesis, and lipid droplet formation. Further mechanistic studies using pharmacological inhibitors revealed that HPIP modulates PRL-induced ß-casein synthesis via phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) activation. This study also identified HPIP as a critical regulator of autocrine PRL signaling as treatment with the PRL receptor antagonist Δ1-9-G129R-hPRL restrained HPIP-mediated PRL synthesis, AKT activation, and ß-casein synthesis in cultured HC11 cells. Interestingly, we also uncovered that microRNA-148a (miR-148a) antagonizes HPIP-mediated mammary epithelial cell differentiation. Together, our study identified HPIP as a critical regulator of PRL signaling and revealed a novel molecular circuitry involving PRL, HPIP, PI3K/AKT, and miR-148a that controls mammary epithelial cell differentiation in vitro.

HPIP protooncogene differentially regulates metabolic adaptation and cell fate in breast cancer cells under glucose stress via AMPK and RNF2 dependent pathways.

While cancer cells rewire metabolic pathways to sustain growth and survival under metabolic stress in solid tumors, the molecular mechanisms underlying these processes remain largely unknown. In this study, cancer cells switched from survival to death during the early to late phases of metabolic stress by employing a novel signaling switch from AMP activated protein kinase (AMPK)-Forkhead box O3 (FOXO3a)-hematopoietic PBX1-interacting protein (HPIP) to the ring finger protein 2 (RNF2)-HPIP-ubiquitin (Ub) pathway. Acute metabolic stress induced proto-oncogene HPIP expression in an AMPK-FOXO3a-dependent manner in breast cancer (BC) cells. HPIP depletion reduced cell survival and tumor formation in mouse xenografts, which was accompanied by diminished intracellular ATP levels and increased apoptosis in BC cells in response to metabolic (glucose) stress. Glutamine flux (13C-labeled) analysis further suggested that HPIP rewired glutamine metabolism by controlling the expression of the solute carrier family 1 member 5 (SLC1A5) and glutaminase (GLS) genes by acting as a coactivator of MYC to ensure cell survival upon glucose deprivation. However, in response to chronic glucose stress, HPIP was ubiquitinated by the E3-Ub ligase, RNF2, and was concomitantly degraded by the proteasome-mediated pathway, ensuring apoptosis. In support of these data, clinical analyses further indicated that elevated levels of HPIP correlated with AMPK activation in BC. Taken together, these data suggest that HPIP is a signal coordinator during metabolic stress and thus serves as a potential therapeutic target in BC.

Two decades of a protooncogene HPIP/PBXIP1: Uncovering the tale from germ cell to cancer.

Hematopoietic PBX interacting protein (HPIP or pre-B-cell leukemia transcription factor interacting protein (PBXIP1) was discovered two decades ago as a corepressor of pre-B-cell leukemia homeobox (PBX) 1 with a vital functional role in hematopoiesis. Later it emerged as a potential biomarker of poor prognosis and tumorigenesis for more than a dozen different cancers. It regulates aggressive cancer phenotypes, cell proliferation, metastasis, EMT, etc. The anomaly in the regulation of HPIP is linked with physiological disorders like renal fibrosis, chronic kidney disease and osteoarthritis. Scientists have unraveled more than twenty interacting proteins of HPIP and its functional role in various physiological and cellular processes that involves normal neuronal development, embryogenesis, endometrium decidualization, and germ cell proliferation. Over the past 20 years, we have witnessed the emerging role of HPIP and its association with a myriad of cellular activities ranging from germ cell proliferation to cancer aggressiveness, modulating multitude of signaling cascades like TGF-ß1, PI3K/AKT, Wnt, mTOR, and Sonic hedgehog signaling pathways. This review will give the current understanding of HPIP, in terms of its diverse functions, theoretical ideas, and further explore cellular links and promising areas that need to be investigated. We also provide a comprehensive overview of the transcript variants of HPIP and distinct sets of transcription factors regulating their expression, which may help to understand the role of HPIP in various cellular or physiological conditions.

Effective in vitro delivery of paclitaxel by nanocargo of mesoporous polycaprolactone against triple negative breast cancer cells by minimalizing drug dose.

Among the breast cancers, triple negative breast cancer (TNBC) has relatively poor outcomes with a lower survival rate and personalised chemotherapy is the only option available for treatment. Currently in the biomedical domain, nanomaterials with porous morphology have revealed their tremendous possibilities to be used as a nanocarrier in treating cancer by offering void space to encapsulate/entrap biological agents. However, the development of nanocarrier-based targeted therapy with high therapeutic efficacy and fewer side effects to normal cells is always a challenge. Here, we have developed nanocargos based on biodegradable mesoporous PCL (polycaprolactone) of approx. diameter of 75 nm by template removal synthesis techniques. Succeeding the comparative analysis of the nanocarriers, the efficiencies of core shell PCL-mZnO (PZ) and mesoporous PCL (HPZ) to deliver paclitaxel (Taxol/T) into breast cancer cells, is investigated. We found that HPZ nanocapsules have less cytotoxicity and drug loading efficiency of about 600 µg mg-1. The Taxol-loaded nanoparticles (T-HPZ) have exhibited more cytotoxicity than Taxol alone treated cancer cells. Furthermore, T-HPZ treated MDA-MB231 cells are accumulated at G2/M phase of the cell cycle and eventually undergo apoptosis. In support of this, anchorage independent growth of MDA-MB231 cells are significantly inhibited by T-HPZ treatment. Together, our findings suggest that T-HPZ-based paclitaxel (Taxol/T) loaded nanoparticles provide a novel therapeutic option in the treatment of TNBC.

Hematopoietic PBX-interacting protein is a substrate and an inhibitor of the APC/C-Cdc20 complex and regulates mitosis by stabilizing cyclin B1.

Proper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein that plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and immunoblotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G2/M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-Cdc20-mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-Cdc20 that maintains the temporal stability of cyclin B1 during the G2/M transition and thereby controls mitosis and cell division.

Amino acid starvation sensing dampens IL-1ß production by activating riboclustering and autophagy.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1ß (IL-1ß) and provides protection from intestinal inflammation in mice. HF inhibits IL-1ß through general control nonderepressible 2 kinase (GCN2)-dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1ß mRNA from translationally active polysomes to inactive ribocluster complexes-such as stress granules (SGs)-via recruitment of RNA-binding proteins (RBPs) T cell-restricted intracellular antigen-1(TIA-1)/TIA-1-related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1ß production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1ß is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.

A transcriptional repressive role for epithelial-specific ETS factor ELF3 on oestrogen receptor alpha in breast cancer cells.

Oestrogen receptor-α (ERα) is a ligand-dependent transcription factor that primarily mediates oestrogen (E2)-dependent gene transcription required for mammary gland development. Coregulators critically regulate ERα transcription functions by directly interacting with it. In the present study, we report that ELF3, an epithelial-specific ETS transcription factor, acts as a transcriptional repressor of ERα. Co-immunoprecipitation (Co-IP) analysis demonstrated that ELF3 strongly binds to ERα in the absence of E2, but ELF3 dissociation occurs upon E2 treatment in a dose- and time-dependent manner suggesting that E2 negatively influences such interaction. Domain mapping studies further revealed that the ETS (E-twenty six) domain of ELF3 interacts with the DNA binding domain of ERα. Accordingly, ELF3 inhibited ERα's DNA binding activity by preventing receptor dimerization, partly explaining the mechanism by which ELF3 represses ERα transcriptional activity. Ectopic expression of ELF3 decreases ERα transcriptional activity as demonstrated by oestrogen response elements (ERE)-luciferase reporter assay or by endogenous ERα target genes. Conversely ELF3 knockdown increases ERα transcriptional activity. Consistent with these results, ELF3 ectopic expression decreases E2-dependent MCF7 cell proliferation whereas ELF3 knockdown increases it. We also found that E2 induces ELF3 expression in MCF7 cells suggesting a negative feedback regulation of ERα signalling in breast cancer cells. A small peptide sequence of ELF3 derived through functional interaction between ERα and ELF3 could inhibit DNA binding activity of ERα and breast cancer cell growth. These findings demonstrate that ELF3 is a novel transcriptional repressor of ERα in breast cancer cells. Peptide interaction studies further represent a novel therapeutic option in breast cancer therapy.