Pro-tumor Functions Research Articles

High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis.

Patients with colorectal liver metastasis (CLM) present with heterogenous clinical outcomes and improved classification is needed to ameliorate the therapeutic output. Macrophages (Mϕ) hold promise as prognostic classifiers and therapeutic targets. Here, stemming from a single-cell analysis of mononuclear phagocytes infiltrating human CLM, we identified two Mϕ markers associated with distinct populations with opposite clinical relevance. The invasive margin of CLM was enriched in pro-inflammatory monocyte-derived Mϕ (MoMϕ) expressing the monocytic marker SERPINB2, and a more differentiated population, tumor-associated Mϕ (TAM), expressing glycoprotein nonmetastatic melanoma protein B (GPNMB). SERPINB2+ MoMϕ had an early inflammatory profile, whereas GPNMB+ TAMs were enriched in pathways of matrix degradation, angiogenesis, and lipid metabolism and were found closer to the tumor margin, as confirmed by spatial transcriptomics on CLM specimens. In a cohort of patients, a high infiltration of SERPINB2+ cells independently associated with longer disease-free survival (DFS; P = 0.033), whereas a high density of GPNMB+ cells correlated with shorter DFS (P = 0.012) and overall survival (P = 0.002). Cell-cell interaction analysis defined opposing roles for MoMϕ and TAMs, suggesting that SERPINB2+ and GPNMB+ cells are discrete populations of Mϕ and may be exploited for further translation to an immune-based stratification tool. This study provides evidence of how multi-omics approaches can identify nonredundant, clinically relevant markers for further translation to immune-based patient stratification tools and therapeutic targets. GPNMB has been shown to set Mϕ in an immunosuppressive mode. Our high dimensional analyses provide further evidence that GPNMB is a negative prognostic indicator and a potential player in the protumor function of Mϕ populations.

New insights into the role of adipocytes in pancreatic cancer progression: paving the way towards novel therapeutic targets.

Pancreatic cancer (PC) remains one of the most lethal malignancies across the world, which is due to delayed diagnosis and resistance to current therapies. The interactions between pancreatic tumor cells and their tumor microenvironment (TME) allow cancer cells to escape from anti-cancer therapies, leading to difficulties in treating PC. With endocrine function and lipid storage capacity, adipose tissue can maintain energy homeostasis. Direct or indirect interaction between adipocytes and PC cells leads to adipocyte dysfunction characterized by morphological change, fat loss, abnormal adipokine secretion, and fibroblast-like transformation. Various adipokines released from dysfunctional adipocytes have been reported to promote proliferation, invasion, metastasis, stemness, and chemoresistance of PC cells via different mechanisms. Additional lipid outflow from adipocytes can be taken into the TME and thus alter the metabolism in PC cells and surrounding stromal cells. Besides, the trans-differentiation potential enables adipocytes to turn into various cell types, which may give rise to an inflammatory response as well as extracellular matrix reorganization to modulate tumor burden. Understanding the molecular basis behind the protumor functions of adipocytes in PC may offer new therapeutic targets.

Effect of M2-like macrophages of the injured-kidney cortex on kidney cancer progression

Chronic kidney disease (CKD) affects kidney cancer patients’ mortality. However, the underlying mechanism remains unknown. M2-like macrophages have pro-tumor functions, also exist in injured kidney, and promote kidney fibrosis. Thus, it is suspected that M2-like macrophages in injured kidney induce the pro-tumor microenvironment leading to kidney cancer progression. We found that M2-like macrophages present in the injured kidney promoted kidney cancer progression and induced resistance to anti-PD1 antibody through its pro-tumor function and inhibition of CD8+ T cell infiltration. RNA-seq revealed Slc7a11 was upregulated in M2-like macrophages. Inhibition of Slc7a11 with sulfasalazine inhibited the pro-tumor function of M2-like macrophages and synergized with anti-PD1 antibody. Moreover, SLC7A11-positive macrophages were associated with poor prognosis among kidney cancer patients. Collectively, this study dissects the characteristic microenvironment in the injured kidney that contributed to kidney cancer progression and anti-PD1 antibody resistance. This insight offers promising combination therapy with anti-PD1 antibody and macrophage targeted therapy.

Interferon-gamma signaling promotes melanoma progression and metastasis.

Interferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we provide in vivo evidence that treatment of mouse melanoma cell lines with Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, suggesting an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are partially dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment.

Activated NK cells reprogram MDSCs via NKG2D-NKG2DL and IFN-γ to modulate antitumor T-cell response after cryo-thermal therapy

BackgroundMyeloid‐derived suppressor cells (MDSCs) can potently inhibit T-cell activity, promote growth and metastasis of tumor and contribute to resistance to immunotherapy. Targeting MDSCs to alleviate their protumor functions and immunosuppressive...

Abstract A012: Repurposing of the macrolide antibiotic clarithromycin for the prevention of lung cancer

Abstract Lung adenocarcinoma (LUAD), particularly K-ras mutant LUAD, is a leading cause of cancer mortality. Therefore, strategies to prevent K-ras-mutant LUAD in its earliest stages in high-risk individuals (e.g., smokers) are urgently needed to reduce the public burden of this fatal disease. We and others have shown that K-ras driven tumorigenesis in lung is intimately linked to chronic inflammation and ultimately, tumor cells immune-escape. The antibiotic clarithromycin (CAM) was identified as one of the most promising candidates for repurposing with demonstrated immunomodulatory and anticancer properties. CAM is widely used and belongs to the macrolide class of antibiotics which are among the safest broad spectrum antimicrobials available. Abundant preclinical and clinical evidence exists demonstrating the in vitro and in vivo anticancer effects of CAM. It has been shown that macrolide antibiotics exert suppression of inflammation without overt immunosuppressive effects mostly through the inhibition of proinflammatory cytokines in vitro and in vivo. In these studies, we tested the lung cancer prevention efficacy of CAM using a Kras mutant lung cancer model. In the CCSPCre; LSL-Kras- G12 D (CC-LR) model, activation of the KrasG12 D mutation takes place in club cells by means of removal of the lox-stop-lox genomic sequence via expression of Cre recombinase under the control of the CCSP promoter. This model is excellent for reproducing the various premalignant to malignant progression steps in the lung. CC-LR mice of both genders were randomly enrolled to four experimental arms comparing three CAM doses: 10mg/kg/day, 50mg/kg/day, and 100mg/kg/day, to vehicle control (H2O). Treatment was administered by oral gavage, 5 times per week for 10 wks., starting at 4 wks. of age. At 14 wks. of age mice were euthanized, lung surface tumors were counted, bronchial lavage fluid, as well as lung samples, were obtained for histological, immunohistochemical (IHC) and qRT-PCR analyses. Clarithromycin treatment led to significant lung cancer prevention efficacy, as determined by lung surface tumor counts. A clear dose response with CAM was observed with a mean lung surface tumor count of 30.2 tumors per mouse for vehicle (n=12 mice), 23.5 for CAM 10mg/kg (n=8), 18.8 for CAM 50mg/kg (n=8) and 13.5 tumors per mouse for the CAM 100mg/kg treatment group (n=12, p=0.0014). A significant decrease in the incidence and multiplicity of premalignant and malignant lesions was also observed in histological analyses. For profiling of the lung immune microenvironment, we analyzed by qRT-PCR the expression of 18 genes identifying various cytokines, cell surface markers and proteins characteristic of specific activation states on the various cell subtypes in the tumor microenvironment. We found significant reduction in the expression of pro-inflammatory cytokines, IL-6, TNF, and IL-1β that are known to have a pro-tumor function in lung tumorigenesis, shown previously by us and other groups. We also found a reduction in the expression of myeloid cell-specific immunosuppressive markers, Arg1 and Fizz 1, that could be due to the reduction in the Gr1+ myeloid cell population, which was detected by means of IHC analyses on the same samples, or due to reprograming of pro-tumor M2 type macrophages toward an anti-tumor M1 phenotype. We also see a trend toward reduction of IL-17 cytokine, which we have previously shown to have an essential role in the promotion of K-ras mutant lung tumors. This was associated with an increase, although not significant, in the expression of anti-tumor Th1-specific transcription factor. However, we see an increase in the expression of PD-1, which could be due to the potential induction of an exhausted T cell phenotype. Taken together, we see a reprograming of the lung microenvironment from a tumor-promoting immunosuppressive phenotype toward an anti-tumor phenotype. This work was supported by NCI PREVENT TORFP75N91019F00131 (TORFP 2019 E-05). Citation Format: Seyed Javad Moghaddam, Tabish Hussain, Melody Zarghooni, Walter V. Velasco, Linda Phan, Michelle I. Savage, Jennifer T. Fox, Shizuko Sei, Powel H. Brown, C. Marcelo Aldaz. Repurposing of the macrolide antibiotic clarithromycin for the prevention of lung cancer [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A012.

Combined single-cell RNA-seq and bulk RNA-seq to analyze the expression and role of TREM2 in bladder cancer.

Currently, reprogramming macrophages has emerged as one of the most promising therapeutic strategies in cancer treatment. Many studies have found that myeloid trigger receptor-2 (TREM2) is mainly expressed on tumor-associated macrophages (TAMs), and targeting TREM2 promotes reprogramming of TAMs and enhances the immunotherapeutic effect of tumors. Nevertheless, the expression and role of TREM2 in different tumor tissues are still controversial. For example, some studies have found that TREM2 can also be expressed on tumor cells and exert pro-tumor functions. It has also been found that TREM2 expression can inhibit tumorigenesis and progression. In fact, there are still no relevant studies on the expression and role of TREM2 in bladder cancer (BLCA). Therefore, the present study combined single-cell RNA-seq and bulk RNA-seq to analyze the expression, role, and molecular mechanism of TREM2 in BLCA. We found that TREM2 was predominantly expressed on TAMs in BLCA, followed by tumor epithelial cells. This finding could be useful for further exploration of the role and mechanism of TREM2. Moreover, TREM2 expression correlates with clinical progression and immunotherapy efficacy, and is an important predictor of prognosis for BLCA patients. Not only that, we also found that TREM2 may exert its effects by promoting epithelial mesenchymal transition (EMT) and T-cell exhaustion. TREM2+ TAMs may play an important pro-tumor role through PTN, ANGPTL, and VISFATIN pathways. In conclusion, our study found that TREM2 is not only a predictor of BLCA prognosis, but also a potential therapeutic target for BLCA.

Abstract C056: The prolyl isomerase PIN1 controls fibroblast state plasticity to impact pancreatic cancer

Abstract Pancreatic cancer associated fibroblasts (CAFs) have been described to play multiple, often conflicting, roles to support or restrict tumor growth. Recent work suggests that heterogeneous differentiation states of fibroblasts contribute to these diverse functions and that reprogramming of fibroblast states can influence tumor outcomes. The impact of distinct fibroblast subpopulations on heterogeneous tumor development and growth, however, remain incompletely understood. PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. PIN1 is overexpressed in cancer and contributes to cancer cell-intrinsic pro-tumorigenic behaviors including cellular proliferation and migration. While its pro-tumor functions have generated interest in therapeutic targeting of PIN1 for cancer treatment, the direct effects of PIN1 inhibition on tumor-associated stromal phenotypes are poorly understood. We have found that PIN1 loss or inhibition results in decreased tumor growth in vivo in transplant or genetically engineered mouse models. Moreover, in syngeneic orthotopic allograft models in which loss of PIN1 is restricted to the tumor host, we observed that KPC tumor cells still have decreased growth, suggesting a critical role for PIN1 in the tumor microenvironment. We observed that PIN1 loss or inhibition in vivo was accompanied by decreased expression of alpha-SMA, a marker of myofibroblast-like CAFs, and have identified a role for PIN1 in the phenotypic and epigenetic response to TGF-beta, a major driver of the myCAF state. PIN1low pancreatic stellate cells or CAFs display altered cell morphology, decreased proliferation, decreased ECM deposition, as well as altered paracrine signaling to cancer cells and other stromal cells. We are currently using 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the molecular mechanisms by which PIN1 controls fibroblast phenotypes and functional impact of altering fibroblast state on tumor phenotypes and outcomes. In addition, we are defining PIN1-dependent mechanisms of crosstalk between neoplastic and non-neoplastic cells and are investigating the requirements for specific fibroblast states to support in vivo growth of heterogeneous pancreatic cancer cells. Citation Format: Ellen M. Langer, Vidhi Shah, Amy Farrell, Colin Daniel, Xiaoyan Wang, Kevin MacPherson, Brittany L. Allen-Petersen, Motoyuki Tsuda, Mara Sherman, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 controls fibroblast state plasticity to impact pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C056.

MEN1703-Mediated PIM Kinases Inhibition Impairs Protumoral and Immunosuppressive Phenotype and Functions of Macrophages in Classical Hodgkin Lymphoma

Increased abundance of tumor-associated macrophages (TAMs) predicts shortened survival of patients with classical Hodgkin lymphoma (cHL). TAMs in cHL might support growth of the malignant, Reed-Sternberg (RS) cells and due to expression of PD-L1, contribute to T-cell exhaustion. In our previous studies, we identified almost universal expression of PIM-1/2/3 kinases (PIMs) in RS cells, but also noted that they are expressed in TAMs. In RS cells, PIMs support malignant cells survival and immune privilege. However, their role in the biology of cHL-TAMs remains to be explored. Herein, using an in vitro model of cHL-TAMs (RS-conditioned macrophages, RS-M) we characterize consequences of PIM inhibition for phenotype and functions of TAMs in cHL. THP1 cells and donor-derived monocytes were differentiated intomacrophages(Mϕ-0) using PMA and CSF-1 respectively. Mϕ-0 were next cocultured with L428 or L1236 RS cells under conditions prohibiting direct contacts. Compared to Mϕ-0, RS-M exhibited elevated expression of PIMs and genes involved in chemotaxis/immunomodulation (CCLs: 2, 5, 7, 8, 13, 17, 18 and 24), extracellular matrix organization (CD206, TGM2 and MMP-1, -7, -9 and -12), T cell exhaustion (PD-L1) and angiogenesis (VEGFA, PDGFB, CHI3L1/2). PIMs expression was also detected in primary cHL-TAMs. To assess role of PIM kinases in TAMs, RS-M were treated with a dual pan-PIM/FLT3 inhibitor, MEN1703, and subjected to transcriptional, biochemical and immunophenotype analyses. MEN1703 skewed gene expression profiles of RS-M toward pro-inflammatory (M1) macrophages and downregulated expression of genes associated with pro-tumoral (M2) macrophages. Consistently, PIM inhibition in RS-M downregulated expression or decreased activity of M2-associated molecules (CREB1, AKT, STAT3/6, CD163, CD206, CD209), and decreased PD-L1 levels by 32%. PIM inhibition hampered important functions of RS-M, including their ability to promote angiogenesis, matrix remodelling (collagen uptake), and eosinophil recruitment. In direct cocultures, RS-M decreased activity of T lymphocytes. This suppressive effect was alleviated in cocultures with the PIM inhibitor-treated RS-M. Our data suggest that PIMs support pro-tumoral and immunosuppressive phenotype of cHL-TAMs. Since PIM activity is required for RS cell survival and immune escape, these kinases are rational targets for therapy in cHL, suggesting the use of PIM inhibitors, such as MEN1703, as a possible therapeutic approach .

Abstract C083: Deconvoluting the metastatic PDA tumor microenvironment

Abstract Pancreatic Ductal Adenocarcinoma (PDA) is the 3rd leading cause of cancer related deaths in the US with a 5-year survival of 11%. Most patients die from disseminated disease, with liver being the most frequently colonized metastatic site. In primary PDA tumors, the tumor microenvironment has been extensively linked to therapy resistance and immune suppression. However, the extent that the microenvironment in the liver metastatic niche promotes immune evasion compared to primary PDA lesions remains unclear. Importantly, strategies to effectively target metastatic disease will provide the most immediate benefit for clinical treatment of PDA. The heterogeneous tumor microenvironment has been recognized as a primary barrier to PDA treatment. Here, the immune and stromal cells are not functioning in a manner beneficial for the host. Numerous cell types have been shown to have a pro-tumor function including regulatory T cells, tumor associated macrophages (TAMs), myeloid derived suppressor cells, and cancer associated fibroblasts (CAFs). Recently, we and others have shown that there are metabolic crosstalk interactions between cancer cells, TAMs, and CAFs. These interactions function to support primary tumor growth, immune suppression, and resistance to chemotherapy. However, the degree that the cancer cell heterogeneity, immune infiltration, and stromal populations present in metastatic lesions mirror primary tumors remains unclear. To directly compare primary vs. metastatic tumors in PDA tumors, we have optimized a syngeneic experimental model to contrast primary vs. metastatic lesions. Using this system, we have identified differential programming among the different cell populations between pancreas and liver PDA lesions using single cell RNA sequencing. Further, we directly compared immune infiltration between primary and metastatic tumors using mass cytometry. Our data indicate alternative mechanisms of immune suppression in liver PDA lesions, which can be directly targeted in combination with checkpoint inhibition strategies. Overall, these data have the potential to lead to new avenues to activate or target the immune system to treat metastatic PDA, an urgent clinical need for pancreatic cancer patients. Citation Format: Rima Singh, Christopher Halbrook, Nina Steele. Deconvoluting the metastatic PDA tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C083.

Abstract PR017: Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is soon to be the second deadliest types of cancer, with a 5-year survival of only 10%. The unique features of PDAC are the expansion of cancer-associated fibroblasts (CAFs), presence of a dense fibrous stroma with high bundled collagen I and immunosuppression, representing a challenge for therapies. Ways to overcome these unique pro-tumor features will be key for the development of better therapeutic strategies for PDAC. Herein we report that, in addition to the identified ectopic expression of NGL-1 in PDAC cells, it is also expressed in the stroma (CAFs and immune cells) of PDAC patients, inversely correlating with overall survival. Stromal NGL-1 was important for tumorigenesis in vivo, as NGL-1 knockout mice (KO) orthotopically allografted with pancreatic cancer cells presented less tumor burden. Further analysis showed that the tumors from KO mice presented more CD8+ T cells and less immunosuppressive cytokines, such as TGFβ. Single cell RNAseq analysis of these tumors showed decreased expression of pro-tumor factors, such as immune checkpoint molecules in T cells and TGFβ related genes across different cellular compartments (epithelial, immune and fibroblasts). In accordance with these results, these tumors presented a limited amount of desmoplastic bundled collagen, suggestive of a TGFβ-deficient environment. In order to further dissect between the NGL-1-dependent contributions of immune vs. local stroma (e.g., CAFs) cells, we generated bone marrow chimeras and performed orthotopic injections to generate tumors. The loss of NGL-1 in each cellular compartment alone failed to phenocopy the full body loss of NGL-1, suggesting that NGL-1 in both immune cells and local stromal are important for tumorigenesis. Functionally, NGL-1 deficient CAFs failed to support the survival of starved PDAC cells in vitro, downregulated important myofibroblastic molecules such as p-smad, and produced less immunosuppressive cytokines, suggesting a role for NGL-1 in key pro-tumor features of CAFs. In fact, the fibroblastic NGL-1 dependent immunomodulatory effects were confirmed with human CD8+ T cells from healthy donors, which lost their cytotoxic profile in the presence of conditioned media (CM) from NGL-1+ CAFs, but were able to keep this profile when exposed to CM from NGL-1 deficient CAFs. Bone marrow derived macrophages from KO mice produced less pro-tumor cytokines and CD8+ T cells lacking NGL-1 proliferated more than those from wild type animals, when stimulated in vitro. Mechanistically, while immune cells and CAFs deficient in NGL-1 are both tumor suppressive, the latter can regain pro-tumor functions in response to TGFβ, explaining the need for a global modulation of NGL-1 expression for an anti-tumor effect. Finally, NGL-1 KO mice orthotopically allografted with PDAC cells responded better (smaller tumors) to chemotherapeutical regimen (FOLFIRINOX) compared to WT animals. All these results point to NGL-1 as a potential new target to modulate immunosuppression and tumorigenesis in pancreatic cancer. Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Esteban Martinez, Stephen Sykes, Nina Steele, Marina Pasca di Magliano, Dmitry Zhigarev, Charline Ogier, Huamin Wang, Igor Astsaturov, Kerry Campbell, Edna Cukierman. Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR017.

Tailoring biomaterials and applications targeting tumor-associated macrophages in cancers.

Tumor-associated macrophages (TAMs) play a critical role in supporting tumor growth and metastasis, taming host immunosurveillance, and augmenting therapeutic resistance. As the current treatment paradigms for cancers are generally insufficient to exterminate cancer cells, anti-cancer therapeutic strategies targeting TAMs have been developed. Since TAMs are highly heterogeneous and the pro-tumoral functions are mediated by phenotypes with canonical surface markers, TAM-associated materials exert anti-tumor functions by either inhibiting polarization to the pro-tumoral phenotype or decreasing the abundance of TAMs. Furthermore, TAMs in association with the immunosuppressive tumor microenvironment (TME) and tumor immunity have been extensively exploited in mounting evidence, and could act as carriers or accessory cells of anti-tumor biomaterials. Recently, a variety of TAM-based materials with the capacity to target and eliminate cancer cells have been increasingly developed for basic research and clinical practice. As various TAM-based biomaterials, including antibodies, nanoparticles, RNAs, etc., have been shown to have potential anti-tumor effects reversing the TME, in this review, we systematically summarize the current studies to fully interpret the specific properties and various effects of TAM-related biomaterials, highlighting the potential clinical applications of targeting the crosstalk among TAMs, tumor cells, and immune cells in anti-cancer therapy.

P044: PIM kinases support protumoral and immunosuppressive phenotype and functions of macrophages in classical Hodgkin Lymphoma

P044: PIM kinases support protumoral and immunosuppressive phenotype and functions of macrophages in classical Hodgkin Lymphoma

Aurora A–mediated pyruvate kinase M2 phosphorylation promotes biosynthesis with glycolytic metabolites and tumor cell cycle progression

Cancer cells have distinctive demands for intermediates from glucose metabolism for biosynthesis and energy in different cell cycle phases. However, how cell cycle regulators and glycolytic enzymes coordinate to orchestrate the essential metabolic processes are still poorly characterized. Here, we report a novel interaction between the mitotic kinase, Aurora A, and the glycolytic enzyme, pyruvate kinase M2 (PKM2), in the interphase of the cell cycle. We found Aurora A-mediated phosphorylation of PKM2 at threonine 45. This phosphorylation significantly attenuated PKM2 enzymatic activity by reducing its tetramerization and also promoted glycolytic flux and the branching anabolic pathways. Replacing the endogenous PKM2 with a nonphosphorylated PKM2 T45A mutant inhibited glycolysis, glycolytic branching pathways, and tumor growth in both invitro and invivo models. Together, our study revealed a new protumor function of Aurora A through modulating a rate-limiting glycolytic enzyme, PKM2, mainly during the S phase of the cell cycle. Our findings also showed that although both Aurora A and Aurora B kinase phosphorylate PKM2 at the same residue, the spatial and temporal regulations of the specific kinase and PKM2 interaction are context dependent, indicating intricate interconnectivity between cell cycle and glycolytic regulators.

Increased glucose metabolism in TAMs fuels O-GlcNAcylation of lysosomal Cathepsin B to promote cancer metastasis and chemoresistance.

How glucose metabolism remodels pro-tumor functions of tumor-associated macrophages (TAMs) needs further investigation. Here we show that M2-like TAMs bear the highest individual capacity to take up intratumoral glucose. Their increased glucose uptake fuels hexosamine biosynthetic pathway-dependent O-GlcNAcylation to promote cancer metastasis and chemoresistance. Glucose metabolism promotes O-GlcNAcylation of the lysosome-encapsulated protease Cathepsin B at serine 210, mediated by lysosome-localized O-GlcNAc transferase (OGT), elevating mature Cathepsin B in macrophages and its secretion in the tumor microenvironment (TME). Loss of OGT in macrophages reduces O-GlcNAcylation and mature Cathepsin B in the TME and disrupts cancer metastasis and chemoresistance. Human TAMs with high OGT are positively correlated with Cathepsin B expression, and both levels predict chemotherapy response and prognosis of individuals with cancer. Our study reports the biological and potential clinical significance of glucose metabolism in tumor-promoting TAMs and reveals insights into the underlying mechanisms.

A calpain-6/YAP axis in sarcoma stem cells that drives the outgrowth of tumors and metastases

Sarcomas include cancer stem cells, but how these cells contribute to local and metastatic relapse is largely unknown. We previously showed the pro-tumor functions of calpain-6 in sarcoma stem cells. Here, we use an osteosarcoma cell model, osteosarcoma tissues and transcriptomic data from human tumors to study gene patterns associated with calpain-6 expression or suppression. Calpain-6 modulates the expression of Hippo pathway genes and stabilizes the hippo effector YAP. It also modulates the vesicular trafficking of β-catenin degradation complexes. Calpain-6 expression is associated with genes of the G2M phase of the cell cycle, supports G2M-related YAP activities and up-regulated genes controlling mitosis in sarcoma stem cells and tissues. In mouse models of bone sarcoma, most tumor cells expressed calpain-6 during the early steps of tumor out-growth. YAP inhibition prevented the neoformation of primary tumors and metastases but had no effect on already developed tumors. It could even accelerate lung metastasis associated with large bone tumors by affecting tumor-associated inflammation in the host tissues. Our results highlight a specific mechanism involving YAP transcriptional activity in cancer stem cells that is crucial during the early steps of tumor and metastasis outgrowth and that could be targeted to prevent sarcoma relapse.

Glucose Metabolism in TAMs Promotes Metastasis and Drug Resistance.

M2-like tumor-associated macrophages (TAM) are addicted to glucose, which promotes their protumor function.

Upregulated Apelin Signaling in Pancreatic Cancer Activates Oncogenic Signaling Pathways to Promote Tumor Development.

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated β-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.

GREM1 is a novel serum diagnostic marker and potential therapeutic target for pancreatic ductal adenocarcinoma.

ObjectivePancreatic ductal adenocarcinoma (PDAC) is a highly malignant neoplasm with rising incidence worldwide. Gremlin 1 (GREM1), a regulator of bone morphogenetic protein (BMP) signaling, fine-tunes extensive biological processes, including organ morphology, cellular metabolism, and multiple pathological developments. The roles of GREM1 in PDAC remain unknown.MethodsVarieties of public databases and online software were employed to analyze the expressions at transcription and protein levels of GREM1 in multiple malignant neoplasms including PDAC, and in addition, its potential pro-tumoral functions in PDAC were further evaluated. A total of 340 serum samples of pancreatic disease, including PDAC, low-grade malignant pancreatic neoplasm, benign pancreatic neoplasm, pancreatitis, and 132 healthy controls, were collected to detect GREM1. The roles of serum GREM1 in the diagnosis and prediction of survival of PDAC after radical resection were also analyzed.ResultsBioinformatics analyses revealed that GREM1 was overexpressed in PDAC and predicted a poorer survival in PDAC. A higher protein level of GREM1 in PDAC correlated with stroma formation and immunosuppression by recruiting varieties of immunosuppressive cells, including T regulatory cells (Tregs), M2 macrophages, myeloid-derived suppressor cells (MDSCs), and exhaustion T cells into the tumor microenvironment. A higher level of serum GREM1 was observed in PDAC patients, compared to healthy control (p < 0.001). Serum GREM1 had a good diagnostic value (area under the curve (AUC) = 0.718, p < 0.001), and its combination with carbohydrate antigen 199 (CA199) achieved a better diagnostic efficacy (AUC = 0.914, p < 0.001), compared to CA199 alone. The cutoff value was calculated by receiver operating characteristic (ROC) analysis, and PDAC patients were divided into two groups of low and high GREM1. Logistic analyses showed serum GREM1 positively correlated with tumor size (hazard ratio (HR) = 7.097, p = 0.032) and histopathological grades (HR = 2.898, p = 0.014). High-level serum GREM1 (1,117.8 pg/ml) showed a shorter postoperative survival (p = 0.0394).ConclusionHigher intra-tumoral expression of GREM1 in PDAC contributes to tumor stroma and immunosuppressive tumor microenvironment, presenting its therapeutic potential. High-level serum GREM1 predicts poorer survival after resection. A combination of serum CA199 and GREM1 shows a stronger diagnostic efficacy in PDAC.

YAP1 acts as a negative regulator of pro-tumor TAZ expression in esophageal squamous cell carcinoma

PurposeAlthough YAP1 and TAZ are believed to be equivalent downstream effectors of the Hippo pathway, differential expression of YAP1 or TAZ suggests distinct functions during cancer progression. The exact role of YAP1 and TAZ in esophageal cancer, the 6th leading cancer-related mortality in the world, remains elusive.MethodsFollowing single or double manipulation of YAP1 or TAZ expression, we subjected these manipulated cells to proliferation, migration, invasion, and xenograft tumorigenesis assays. We used RT-qPCR and Western blotting to examine their expression in the manipulated cells with or without inhibition of transcription or translation. We also examined the impact of YAP1 or TAZ deregulation on clinical outcome of esophageal cancer patients from the TCGA database.ResultsWe found that YAP1 functions as a tumor suppressor whereas TAZ exerts pro-tumor functions in esophageal cancer cells. We also found a significant increase in TAZ mRNA expression upon YAP1 depletion, but not vice versa, despite the downregulation of CTGF and CYR61, shared targets of YAP1 and TAZ, in xenografted tissue cells. In addition to transcriptional regulation, YAP1-mediated TAZ expression was found to occur via protein synthesis. Restored TAZ expression mitigated YAP1-mediated suppression of cellular behavior. By contrast, TAZ silencing reduced the promoting effect exerted by YAP1 depletion on cellular behaviors. The observed anti-tumor function of YAP1 was further supported by a better overall survival among esophageal cancer patients with a high YAP1 expression.ConclusionFrom our data we conclude that YAP1 functions as a suppressor and negatively regulates pro-tumor TAZ expression via transcriptional and translational control in esophageal cancer.