Chimeric galectin-3 and collagens: Biomarkers and potential therapeutic targets in fibroproliferative diseases.

Fibrosis, stiffening and scarring of an organ/tissue due to genetic abnormalities, environmental factors, infection, and/or injury, is responsible for > 40% of all deaths in the industrialized world, and to date, there is no cure for it despite extensive research and numerous clinical trials. Several biomarkers have been identified, but no effective therapeutic targets are available. Human galectin-3 is a chimeric gene product formed by the fusion of the internal domain of the collagen alpha gene [N-terminal domain (ND)] at the 5'-end of galectin-1 [C-terminal domain (CRD)] that appeared during evolution together with vertebrates. Due to the overlapping structural similarities between collagen and galectin-3 and their shared susceptibility to cleavage by matrix metalloproteases to generate circulating collagen-like peptides, this review will discuss present knowledge on the role of collagen and galectin-3 as biomarkers of fibrosis. We will also highlight the need for transformative approaches targeting both the ND and CRD domains of galectin-3, since glycoconjugate binding by the CRD is triggered by ND-mediated oligomerization and the therapies targeted only at the CRD have so far achieved limited success.

MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.

The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.

Contextual reprogramming of CAR-T cells for treatment of HER2+ cancers.

BACKGROUND: Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. METHODS: To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. RESULTS: Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product. CONCLUSIONS: As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

Galectin-3: an immune checkpoint target for musculoskeletal tumor patients.

In the past decade, the development of immune checkpoint inhibitors in oncological clinical settings was in the forefront. However, the interest in musculoskeletal tumor patients as candidates for checkpoint inhibition remains underserved. Here, we are forwarding evidence proposing that galectin-3 (Gal-3) is an additional immune factor in the checkpoint processes. This review is the result of a large-scale cohort study depicting that overexpression of Gal-3 was widely prevalent in patients with musculoskeletal tumors, whereas T cell infiltrations were generally suppressed in the tumor microenvironment. Targeting Gal-3 would serve as a novel immune checkpoint inhibitor candidate in patients afflicted with aggressive musculoskeletal tumors.

CRISPR technology for immuno-oncology applications.

Emergency of the CRISPR technology, a new genome editing tool, revolutionized the biomedical research field in the past 6 years. At the same time, recent advances in cancer immunotherapy reinvigorated our hope to cure most if not all cancer patients with further development of various treatment options. A combination of the CRISPR technology with immuno-oncology research will undoubtedly accelerate the development of new cancer therapies. This review will focus on the CRISPR system and its applications in immune-oncology including identification of immune-oncology gene targets, generation of cancer animal models, and enabling better cell design and manufacture for adoptive cellular therapies.

The CRISPR System and Cancer Immunotherapy Biomarkers.

Recent advances in cancer immunotherapy have shed new light on the possibility to cure most, if not all, cancer patients with further development of various treatment options. The emergency of a new genome editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR) technology, revolutionized the biomedical research field. We envision application of the CRISPR technology in cancer research, diagnosis, and therapy will markedly speed up the development of new treatment options for cancer patients. The CRISPR system and its applications in biomedical research will be discussed with an emphasis on cancer immunotherapy and biomarker development.

p16(Ink4a) and senescence-associated ß-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli.

Constitutive p16Ink4a expression, along with senescence-associated ß-galactosidase (SAßG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAßG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAßG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAßG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAßG-positive macrophages.

Murine mesenchymal cells that express elevated levels of the CDK inhibitor p16(Ink4a) in vivo are not necessarily senescent.

Age-related health decline has been attributed to the accumulation of senescent cells recognized in vivo by p16(Ink4a) expression. The pharmacological elimination of p16(Ink4a)-positive cells from the tissues of mice was shown to extend a healthy lifespan. Here, we describe a population of mesenchymal cells isolated from mice that are highly p16(INK4a)-positive are proficient in proliferation but lack other properties of cellular senescence. These data, along with earlier reports on p16(Ink4a)-positive macrophages, indicate that p16(Ink4a)-positive and senescent cell populations only partially intersect, therefore, extending the list of potential cellular targets for anti- aging therapies.

Aging of mice is associated with p16(Ink4a)- and ß-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells.

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and ß-galactosidase activity at pH6.0 (ß-gal(pH6)), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/ß-gal(pH6)-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/ß-gal(pH6)-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/ß-gal(pH6)-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/ß-gal(pH6)-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Extracellular galectin-3 programs multidrug resistance through Na+/K+-ATPase and P-glycoprotein signaling.

Galectin-3 (Gal-3, LGALS3) is a pleotropic versatile, 29-35 kDa chimeric gene product, and involved in diverse physiological and pathological processes, including cell growth, homeostasis, apoptosis, pre-mRNA splicing, cell-cell and cell-matrix adhesion, cellular polarity, motility, adhesion, activation, differentiation, transformation, signaling, regulation of innate/adaptive immunity, and angiogenesis. In multiple diseases, it was found that the level of circulating Gal-3 is markedly elevated, suggesting that Gal-3-dependent function is mediated by specific interaction with yet an unknown ubiquitous cell-surface protein. Recently, we showed that Gal-3 attenuated drug-induced apoptosis, which is one of the mechanisms underlying multidrug resistance (MDR). Here, we document that MDR could be mediated by Gal-3 interaction with the house-keeping gene product e.g., Na+/K+-ATPase, and P-glycoprotein (P-gp). Gal-3 interacts with Na+/K+-ATPase and induces the phosphorylation of P-gp. We also find that Gal-3 binds P-gp and enhances its ATPase activity. Furthermore Gal-3 antagonist suppresses this interaction and results in a decrease of the phosphorylation and the ATPase activity of P-gp, leading to an increased sensitivity to doxorubicin-mediated cell death. Taken together, these findings may explain the reported roles of Gal-3 in diverse diseases and suggest that a combined therapy of inhibitors of Na+/K+-ATPase and Gal-3, and a disease specific drug(s) might be superior to a single therapeutic modality.

Galectin-3 leads to attenuation of apoptosis through Bax heterodimerization in human thyroid carcinoma cells.

Cancer cells survive escaping normal apoptosis and the blocks in apoptosis that keep cancer cells alive are promising candidates for targeted therapy. Galectin-3 (Gal-3) is, a member of the lectin family, which is involved in cell growth, adhesion, proliferation and apoptosis. It remains elusive to understand the role of Gal-3 on apoptosis in thyroid carcinoma cells. Here, we report that Gal-3 heterodimerizes Bax, mediated by the carbohydrate recognition domain (CRD) of Gal-3, leading to anti-apoptotic characteristic. Gal-3/Bax interaction was suppressed by an antagonist of Gal-3, in which in turn cells became sensitive to apoptosis. The data presented here highlight that Gal-3 is involved in the anti-apoptosis of thyroid carcinoma cells. Thus, it suggests that targeting Gal-3 may lead to an improved therapeutic modality for thyroid cancer.

Galectin-3 induces cell migration via a calcium-sensitive MAPK/ERK1/2 pathway.

The presence and level of circulating galectin-3 (Gal-3), a member of the galectin family, is associated with diverse diseases ranging from heart failure, immune disorders to cancer metastasis and serves as a biomarker of diagnosis and treatment response. However, the mechanisms by which exogenous Gal-3 affects pathobiology events remain elusive. In the current study, we found that exogenous Gal-3 slightly delays, while prolonging tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in HeLa cells through a calcium-sensitive and PKC-dependent signaling pathway. The activation was dependent on the sugar-binding properties of Gal-3, since the antagonist lactose could inhibit it. The sugar-binding motif of Gal-3 was required for the activation of ERK1/2. The activation of ERK1/2 was necessary for the initiation and induction of cell migration associated with the phosphorylation of paxillin. All the results presented in this study suggest a novel calcium-sensitive and PKC-dependent pathway through which circulating Gal-3 promotes cell migration and activating the ERK1/2. Taken together, the data depicted here propose a biological function and a target for the diseases' associated circulating Gal-3.

Autocrine motility factor modulates EGF-mediated invasion signaling.

Autocrine motility factor (AMF) enhances invasion by breast cancer cells, but how its secretion and effector signaling are controlled in the tumor microenvironment is not fully understood. In this study, we investigated these issues with a chimeric AMF that is secreted at high levels through a canonical endoplasmic reticulum (ER)/Golgi pathway. Using this tool, we found that AMF enhances tumor cell motility by activating AKT/ERK, altering actin organization, and stimulating ß-catenin/TCF and activating protein 1 transcription. EGF enhanced secretion of AMF through its casein kinase II-mediated phosphorylation. RNA interference-mediated attenuation of AMF expression inhibited EGF-induced invasion by suppressing extracellular signal-regulated kinase signaling. Conversely, exogenous AMF overcame the inhibitory effect of EGF receptor inhibitor gefitinib on invasive motility by activating HER2 signaling. Taken together, our findings show how AMF modulates EGF-induced invasion while affecting acquired resistance to cytotoxic drugs in the tumor microenvironment.

Galectin-3: a possible complementary marker to the PSA blood test.

The prostate-specific antigen (PSA) test has served as a blood marker of prostate cancer (PCa), and for monitoring recurrence/metastasis in patients after therapeutic intervention. However, the applicability/reliability of the PSA test was recently questioned as it is not without challenges, in particular in men who have PCa without an elevated PSA (false negative), or in men who are disease-free with elevated levels of PSA (false positive). Galectin-3 is a tumor-associated protein; present in the seminal fluid and is a substrate for the PSA enzyme e.g., a chymotrypsin-like serine protease. We hypothesized that the cleavage status and level of galectin-3 in the prostate tissue and sera are associated with PCa. Thus, we compared galectin-3 levels obtained from sera of non-cancer urology patients to those of metastatic PCa patients. The data were confirmed by analyzing PCa tissue arrays. Here, we report that galectin-3 levels in the sera of patients with metastatic PCa were uniformly higher as compared to the non-cancer patient controls. The data suggest that galectin-3 serum level may be a useful serum complementary marker to the PSA blood test to be used for initial and follow-up PSA complimentary diagnostic/prognostic tool for recurrence in PCa patients.

Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking.

Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus-cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3-nucleus translocation rate. Overall, the results show Nup98's involvement in nuclear-cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

Ocimum gratissimum retards breast cancer growth and progression and is a natural inhibitor of matrix metalloproteases.

Ocimum genus (a.k.a holy basil or tulsi) is a dietary herb used for its multiple beneficial pharmacologic properties including anti-cancer activity. Here we show that crude extract of Ocimum gratissimum (OG) and its hydrophobic and hydrophilic fractions (HB and HL) differentially inhibit breast cancer cell chemotaxis and chemoinvasion in vitro and retard tumor growth and temporal progression of MCF10ADCIS.com xenografts, a model of human breast comedo-ductal carcinoma in situ (comedo-DCIS). OG-induced inhibition of tumor growth was associated with decreases in basement membrane disintegration, angiogenesis and MMP-2 and MMP-9 activities as confirmed by in situ gelatin zymography and cleavage of galectin-3. There was also decrease in MMP-2 and MMP-9 activities in the conditioned media of OG-treated MCF10AT1 and MCF10AT1-EIII8 premalignant human breast cancer cells as compared with control. The MMP-2 and MMP-9 inhibitory activities of OG were verified in vitro using gelatin, a synthetic fluorogenic peptide and recombinant galectin-3 as MMP substrates. Mice fed on OG-supplemented drinking water showed no adverse effects compared with control. These data suggest that OG is non-toxic and that the anti-cancer therapeutic activity of OG may in part be contributed by its MMP inhibitory activity.

MEK-1 activates C-Raf through a Ras-independent mechanism.

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using (32)P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating the C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the down-regulation of RKIP and MST2.

Novel inhibitors of Rad6 ubiquitin conjugating enzyme: design, synthesis, identification, and functional characterization.

Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2-M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes ß-catenin, MDA-MB-231 treatment with SMIs #8 or 9 decreased ß-catenin protein levels. Together these results describe identification of the first Rad6 SMIs.

Autocrine motility factor promotes HER2 cleavage and signaling in breast cancer cells.

Trastuzumab (Herceptin) is an effective targeted therapy in HER2-overexpressing human breast carcinoma. However, many HER2-positive patients initially or eventually become resistant to this treatment, so elucidating mechanisms of trastuzumab resistance that emerge in breast carcinoma cells is clinically important. Here, we show that autocrine motility factor (AMF) binds to HER2 and induces cleavage to the ectodomain-deleted and constitutively active form p95HER2. Mechanistic investigations indicated that interaction of AMF with HER2 triggers HER2 phosphorylation and metalloprotease-mediated ectodomain shedding, activating phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase signaling and ablating the ability of trastuzumab to inhibit breast carcinoma cell growth. Furthermore, we found that HER2 expression and AMF secretion were inversely related in breast carcinoma cells. On the basis of this evidence that AMF may contribute to HER2-mediated breast cancer progression, our findings suggest that AMF-HER2 interaction might be a novel target for therapeutic management of patients with breast cancer, whose disease is resistant to trastuzumab.

Galectin-3 binding and metastasis.

Galectin-3 is a member of a family of carbohydrate-binding proteins. It is present in the nucleus, the -cytoplasm, and also the extracellular matrix (ECM) of many normal and neoplastic cell types. Reports show an upregulation of this protein in transformed and metastatic cell lines (Raz and Lotan Cancer Metastasis Rev 6: 433-452, 1987; Raz et al. Int J Cancer 46: 871-877, 1990). Moreover, in many human carcinomas, an increased expression of galectin-3 correlates with progressive tumor stages (Lotan et al. Int J Cancer 56: 474-480, 1994; Bresalier et al. Gastroenterology 115: 287-296, 1998; Nangia-Makker et al. Int J Oncol 7: 1079-1087, 1995; Xu et al. Am J Pathol 147: 815-822, 1995).Several lines of analysis have demonstrated that the galectins participate in cell-cell and cell-matrix interactions by recognizing and binding complementary glycoconjugates and thereby play a crucial role in normal and pathological processes. Elevated expression of the protein is associated with an increased capacity for anchorage-independent growth, homotypic aggregation, and tumor cell lung colonization (Lotan et al. Cancer Res 45: 4349-4353, 1985; Lotan and Raz J Cell Biochem 37: 107-117, 1988; Meromsky et al. Cancer Res 46: 5270-5275, 1986). In this chapter we describe the methods of purification of galectin-3 from transformed Escherichia coli and some of the commonly used functional assays for analyzing galectin-3 binding.