Cancer Cells Promotes Tumor Growth Research Articles

MiR-373-3p promotes aerobic glycolysis in colon cancer cells by targeting MFN2.

MicroRNAs (miRNAs) are implicated in the development of cancers and may serve as potential targets for therapy. However, the functions and underlying mechanisms of miRNAs in cancers are not well understood. This work aims to study the role of miR-373-3p in colon cancer cells. We find that the expression of miR-373-3p mimics promotes and the miR-373-3p inhibitor suppresses aerobic glycolysis and proliferation of colon cancer cells. Mechanistically, miR-373-3p inhibits the expression of MFN2, a gene that is known to suppress glycolysis, which leads to the activation of glycolysis and eventually the proliferation of cells. In a nude mouse tumor model, the expression of miR-373-3p in colon cancer cells promotes tumor growth by enhancing lactate formation, which is inhibited by the co-expression of MFN2 in the cells. Administration of the miR-373-3p antagomir blunts in vivo tumor growth by decreasing lactate production. In addition, in human colon cancers, the expression levels of miR-373-3p are increased, while those of MFN2 mRNA are decreased, and the increase of miR-373-3p is associated with the decrease of MFN2 mRNA. Our results reveal a previously unknown function and underlying mechanism of miR-373-3p in the regulation of glycolysis and proliferation in cancer cells and underscore the potential of targeting miR-373-3p for colon cancer treatment.

Podoplanin promotes tumor growth, platelet aggregation, and venous thrombosis in murine models of ovarian cancer

BackgroundPodoplanin (PDPN) is a sialylated membrane glycoprotein that binds to C‐type lectin‐like receptor 2 on platelets resulting in platelet activation. PDPN is expressed on lymphatic endothelial cells, perivascular fibroblasts/pericytes, cancer cells, cancer‐associated fibroblasts, and tumor stromal cells. PDPN's expression on malignant epithelial cells plays a role in metastasis. Furthermore, the expression of PDPN in brain tumors (high‐grade gliomas) was found to correlate with an increased risk of venous thrombosis. ObjectiveWe examined the expression of PDPN and its role in tumor progression and venous thrombosis in ovarian cancer. MethodsWe used mouse models of ovarian cancer and venous thrombosis. ResultsOvarian cancer cells express PDPN and release PDPN‐rich extracellular vesicles (EVs), and cisplatin and topotecan (chemotherapies commonly used in ovarian cancer) increase the expression of podoplanin in cancer cells. The expression of PDPN in ovarian cancer cells promotes tumor growth in a murine model of ovarian cancer and that knockdown of PDPN gene expression results in smaller primary tumors. Both PDPN‐expressing ovarian cancer cells and their EVs cause platelet aggregation. In a mouse model of venous thrombosis, PDPN‐expressing EVs released from HeyA8 ovarian cancer cells produce more frequent thrombosis than PDPN‐negative EVs derived from PDPN‐knockdown HeyA8 cells. Blood clots induced by PDPN‐positive EVs contain more platelets than those in blood clots induced by PDPN‐negative EVs. ConclusionsIn summary, our findings demonstrate that the expression of PDPN by ovarian cancer cells promotes tumor growth and venous thrombosis in mice.

Abstract 4117: Adipsin promotes tumor progression in ESR1 mutant breast cancer cells

Abstract ESR1 point mutations at ESR1 ligand-binding domain are frequently identified in metastatic tumors, cfDNA (Cell-free DNA), ctDNA (Circulating tumor DNA) and ptDNA (plasma tumor DNA) derived from ESR1 positive breast cancer patients treated SERMs, SERDs, and AIs. ESR1 mutations enhance ESR1 transcriptional activity in the absence of estrogen and induce estradiol independent growth as well as increase resistance to SERM or SERD in ESR1 mutant overexpressed cells. ESR1 mutations are found more frequently in cfDNA and tumor DNA from patients with metastatic disease compared to patients with primary tumor, Although SERM, SERD, AI, and CDK4/6 inhibitor therapies have demonstrated preclinical and clinical benefits for breast cancer with ESR1 mutations, the development of resistance remains a significant challenge and the detailed mechanisms and potential therapeutic targets in metastatic breast cancer with ESR1 point mutations is yet to be revealed. Tumor and organ microenvironments are crucial for cancer progression and metastasis. Crosstalk between multiple non-malignant cell types in the microenvironments such as blood and lymphatic endothelial cells and cancer cells promotes tumor growth and metastasis. Possibly the anti-angiogenic and anti-lymphangiogenic therapies can be combined with each other for improved outcomes for patients. In this study, we identified the secretion of Adipsin from two different genome-edited MCF-7 harboring Y537S and D538G ESR1 cells compared to wild-type cells using the Human Cytokine Array Q440 to quantitatively detect 440 human inflammatory factors, growth factors, chemokines, receptors, and cytokines simultaneously. We validated that the expression of Adipsin was highly upregulated in MCF-7-Y537S and D538G ESR1 mutant cells by real-time qRT-PCR and ELISA. Interestingly, we observed that when cells were cultured in estrogen deprivation, the mRNA expression of the Adipsin was significantly increased in MCF-7-ESR1 mutant cells while the treatment of tamoxifen and fulvestrant abrogated the upregulation of Adipsin. These results suggest that Adipsin is an ER target gene. Since the cleavage of factor B by Adipsin results in the conversion of C3 to C3a, which binds to its cell surface receptor (C3aR), our data showed that the C3a production was significantly increased in ESR1 mutant cells compared to WT by ELISA. In addition, we found that C3aR expression was increased in ESR1 mutant cells. To elucidate that the C3aR signaling pathway promotes the proliferation of MCF-7-ESR1 mutant cells, we examined the ESR1 mutant cell viability using SB290157, C3aR inhibitor and found that ESR1 mutant cell growth was decreased in the treatment of SB290157. Furthermore, we found that apoptosis was significantly induced in ESR1 mutant cells compared to WT by Annexin V assay. These findings implicate Adipsin signaling as a critical event in breast cancer metastasis with ESR1 mutations. Further, these studies suggest that Adipsin acts as a key regulator orchestrating breast cancer with ESR1 mutations. Therefore, we have provided evidence that supports the hypothesis that functional inhibition of the Adipsin signaling pathway has the potential to circumvent breast cancer metastasis. Citation Format: Marie K. Malone, Kideok Jin. Adipsin promotes tumor progression in ESR1 mutant breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4117.

Inhibition of colorectal cancer-associated fibroblasts by lipid nanocapsules loaded with acriflavine or paclitaxel.

Crosstalk between cancer-associated fibroblasts (CAFs) and colorectal cancer cells promotes tumor growth and contributes to chemoresistance. In this study, we assessed the sensitivity of a primary CAF cell line, CT5.3hTERT, to standard-of-care and alternative cytotoxic treatments. Paclitaxel (PTX) and acriflavine (ACF) were identified as the most promising molecules to inhibit CAF development. To allow the translational use of both drugs, we developed lipid nanocapsule (LNC) formulations for PTX and ACF. Finally, we mixed CAFs and tumor cell lines in a cocultured spheroid, and the effect of both drugs was investigated by histological analyses. We demonstrated CAF inhibition by LNC-ACF and whole tumor inhibition by LNC-PTX. Altogether, we proposed a new strategy to reduce CAF populations in the colorectal microenvironment that should be tested in vivo.

Structural and Physical Basis for the Higher Affinity to Oncoprotein MDM2 of a Peptide Selected with MRNA Display Over Tumor Suppressor p53

Inactivation of the tumor suppressor p53 by oncoprotein MDM2 overexpressed in cancer cells promotes tumor growth. The peptides whose sequence mimic the MDM2-binding region within the intrinsically disordered region of p53 reportedly restore the anti-tumor activities of p53. We previously obtained the 12-mer peptide MIP, which has a higher affinity to MDM2 than the known peptides, such as p53 peptide and DI. MIP exhibits higher potency to prevent tumor cell growth. Here, we determined the NMR structure of the MIP:MDM2 complex.

TGF-βRII Knock-down in Pancreatic Cancer Cells Promotes Tumor Growth and Gemcitabine Resistance. Importance of STAT3 Phosphorylation on S727.

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers in the Western world because of a lack of early diagnostic markers and efficient therapeutics. At the time of diagnosis, more than 80% of patients have metastasis or locally advanced cancer and are therefore not eligible for surgical resection. Pancreatic cancer cells also harbour a high resistance to chemotherapeutic drugs such as gemcitabine that is one of the main palliative treatments for PDAC. Proteins involved in TGF-β signaling pathway (SMAD4 or TGF-βRII) are frequently mutated in PDAC (50–80%). TGF-β signalling pathway plays antagonistic roles during carcinogenesis by initially inhibiting epithelial growth and later promoting the progression of advanced tumors and thus emerged as both tumor suppressor and oncogenic pathways. In order to decipher the role of TGF-β in pancreatic carcinogenesis and chemoresistance, we generated CAPAN-1 and CAPAN-2 cell lines knocked down for TGF-βRII (first actor of TGF-β signaling). The impact on biological properties of these TGF-βRII-KD cells was studied both in vitro and in vivo. We show that TGF-βRII silencing alters tumor growth and migration as well as resistance to gemcitabine. TGF-βRII silencing also leads to S727 STAT3 and S63 c-Jun phosphorylation, decrease of MRP3 and increase of MRP4 ABC transporter expression and induction of a partial EMT phenotype. These markers associated with TGF-β signaling pathways may thus appear as potent therapeutic tools to better treat/manage pancreatic cancer.

Identification of an Unfavorable Immune Signature in Advanced Lung Tumors from Nrf2-Deficient Mice.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway in normal cells inhibits carcinogenesis, whereas constitutive activation of Nrf2 in cancer cells promotes tumor growth and chemoresistance. However, the effects of Nrf2 activation in immune cells during lung carcinogenesis are poorly defined and could either promote or inhibit cancer growth. Our studies were designed to evaluate tumor burden and identify immune cell populations in the lungs of Nrf2 knockout (KO) versus wild-type (WT) mice challenged with vinyl carbamate. Nrf2 KO mice developed lung tumors earlier than the WT mice and exhibited more and larger tumors over time, even at late stages. T cell populations were lower in the lungs of Nrf2 KO mice, whereas tumor-promoting macrophages and myeloid-derived suppressor cells were elevated in the lungs and spleen, respectively, of Nrf2 KO mice relative to WT mice. Moreover, 34 immune response genes were significantly upregulated in tumors from Nrf2 KO mice, especially a series of cytokines (Cxcl1, Csf1, Ccl9, Cxcl12, etc.) and major histocompatibility complex antigens that promote tumor growth. Our studies discovered a novel immune signature, characterized by the infiltration of tumor-promoting immune cells, elevated cytokines, and increased expression of immune response genes in the lungs and tumors of Nrf2 KO mice. A complementary profile was also found in lung cancer patients, supporting the clinical significance of our findings. Overall, our results confirmed a protective role for Nrf2 in late-stage carcinogenesis and, unexpectedly, suggest that activation of Nrf2 in immune cells may be advantageous for preventing or treating lung cancer. Antioxid. Redox Signal.

Abstract B031: Semaphorin 3E promotes pancreatic cancer metastasis through activating stromal cell

Abstract Background: Better understanding of the molecular mechanisms of pancreatic adenocarcinoma (PDAC) metastasis is important for the development of effective treatments for this devastating disease. We have previously shown that overexpression of Semaphorin 3E (Sema3E) enhances PDAC cell growth and is associated with poor patient survival. Here, we further determined the effect of Sema3E on stromal cells and the molecular mechanisms contributing to PDAC metastasis. Materials and Methods: Panels of stable PDAC cell lines were generated using either lenti-Sema3E for overexpression or the Crispr/Cas9 system to knock out (KO) Sema3E in both panc28 and panc48 cell lines. The effects of Sema3E overexpression or KO were measured in orthotopically implanted xenograft tumor mouse models in respect to tumor size, metastasis, and survival. After coculture with pancreatic stellate cells (PSCs), the effects of Sema3E on PSC activation were evaluated by GFAP and αSMA staining. Mixtures of PDAC cell lines with PSCs in vivo tumor growth were also evaluated. Result: After orthotopically implanting a panel of Sema3E-overexpressing or knockout panc28 cell lines, we found that the survival span of the sema3E-overexpression cell implant group was significantly shorter than the vector control group (31±9 days vs. 51±15 days) (p<0.05). In addition, more lung metastases were found in the Panc28-Sema3E overexpressing group. Compared to the primary tumor, the lung metastases had increased sema3E expression. Furthermore, Sema3E overexpression promoted PSC activation with a greater ratio of αSMA to GFAP staining when cocultured with PSC cells. Cotransplant of Sema3E-overexpression cells and PSC cells promoted tumor growth and metastasis when compare to Sema3E-overexpressed cells alone. Conclusion: Sema3E overexpression in pancreatic cancer cells promotes tumor growth, lung metastasis, and poor survival. The interaction between Sema3E and PSC cells may play an important role in contributing to tumor growth and metastasis. Citation Format: Zhengdong Liang, Lin-Kin Yong, George Van Buren, William Fisher, Rosa Hwang, Changyi Chen, Qizhi Cathy Yao. Semaphorin 3E promotes pancreatic cancer metastasis through activating stromal cell [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B031.

Overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating chemokine expression.

We previously found that the scaffold adapter GRB2-associated binding protein 2 (GAB2) is amplified and overexpressed in a subset of primary high-grade serous ovarian cancers and cell lines. Ovarian cancer cells overexpressing GAB2 are dependent on GAB2 for activation of the phosphatidylinositol 3-kinase (PI3K) pathway and are sensitive to PI3K inhibition. In this study, we show an important role of GAB2 overexpression in promoting tumor angiogenesis by upregulating expression of multiple chemokines. Specifically, we found that suppression of GAB2 by inducible small hairpin RNA in ovarian cancer cells inhibited tumor cell proliferation, angiogenesis and peritoneal tumor growth in immunodeficient mice. Overexpression of GAB2 upregulated the secretion of several chemokines from ovarian cancer cells, including CXCL1, CXCL2 and CXCL8. The secreted chemokines not only signal through endothelial CXCR2 receptor in a paracrine manner to promote endothelial tube formation, but also act as autocrine growth factors for GAB2-induced transformation of fallopian tube secretory epithelial cells and clonogenic growth of ovarian cancer cells overexpressing GAB2. Pharmacological inhibition of inhibitor of nuclear factor kappa-B kinase subunit β (IKKβ), but not PI3K, mechanistic target of rapamycin (mTOR) or mitogen-activated protein kinase (MEK), could effectively suppress GAB2-induced chemokine expression. Inhibition of IKKβ augmented the efficacy of PI3K/mTOR inhibition in suppressing clonogenic growth of ovarian cancer cells with GAB2 overexpression. Taken together, these findings suggest that overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating expression of CXCL1, CXCL2 and CXCL8 that is IKKβ-dependent. Co-targeting IKKβ and PI3K pathways downstream of GAB2 might be a promising therapeutic strategy for ovarian cancer that overexpresses GAB2.

Activating transcription factor 4 promotes angiogenesis of breast cancer through enhanced macrophage recruitment.

Angiogenesis plays an important role in the progression of tumor. Besides being regulated by tumor cells per se, tumor angiogenesis is also influenced by stromal cells in tumor microenvironment (TME), for example, tumor associated macrophages (TAMs). Activating transcription factor 4 (ATF4), a member of the ATF/CREB family, has been reported to be related to tumor angiogenesis. In this study, we found that exogenous overexpression of ATF4 in mouse breast cancer cells promotes tumor growth via increasing tumor microvascular density. However, ATF4 overexpression failed to increase the expression level of a series of proangiogenic factors including vascular endothelial growth factor A (VEGFA) in tumor cells in this model. Thus, we further investigated the infiltration of proangiogenic macrophages in tumor tissues and found that ATF4-overexpressing tumors could recruit more macrophages via secretion of macrophage colony stimulating factor (M-CSF). Overall, we concluded that exogenous overexpression of ATF4 in breast cancer cells may facilitate the recruitment of macrophages into tumor tissues and promote tumor angiogenesis and tumor growth indirectly.

Mathematical modeling of Interleukin-35 promoting tumor growth and angiogenesis.

Interleukin-35 (IL-35), a cytokine from the Interleukin-12 cytokine family, has been considered as an anti-inflammatory cytokine which promotes tumor progression and tumor immune evasion. It has also been demonstrated that IL-35 is secreted by regulatory T cells. Recent mouse experiments have shown that IL-35 produced by cancer cells promotes tumor growth via enhancing myeloid cell accumulation and angiogenesis, and reducing the infiltration of activated CD8 T cells into tumor microenvironment. In the present paper we develop a mathematical model based on these experimental results. We include in the model an anti-IL-35 drug as treatment. The extended model (with drug) is used to design protocols of anti-IL-35 injections for treatment of cancer. We find that with a fixed total amount of drug, continuous injection has better efficacy than intermittent injections in reducing the tumor load while the treatment is ongoing. We also find that the percentage of tumor reduction under anti-IL-35 treatment improves when the production of IL-35 by cancer is increased.

Abstract 771: TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis

Abstract Background: Paclitaxel (PXL) is a widely-used drug for breast cancer therapy. Resistance, however, occurs frequently and the evasion mechanisms remain unclear. PXL is known to activate Toll-like Receptor-4 (TLR4), a major signaling receptor expressed on immune cells responding to lipopolysaccharide (LPS). LPS activation of TLR4 in the immune cells substantially enhances their migratory, invasive, proliferative, and pro-survival functions as necessary for the body defense. Since PXL is a functional LPS mimetic, we hypothesized that acquisition of such phenotype by TLR4-expressing cancer cells promotes tumor growth, metastasis, and resistance to therapy. Methods: We tested this hypothesis in two breast cancer luciferase-tagged models genetically modified to either suppress TLR4 expression in a positive line, MDA-MB-231, or overexpress it in a negative line, HCC1806. Modified cell lines and their controls were orthotopically implanted in mice followed by measuring tumor growth prior and post-PXL treatment. Incidence and burden of lymph node (LN) and lung metastases were quantified by measuring luciferase activity in respective organs. TLR4-induced inflammation was assessed by measuring cytokines in blood, tumor lysates, and metastatic organs using RT-qPCR and ELISA. Cell composition of spleens and bone marrow from control and treated mice were analyzed by FACS. TLR4-induced changes in tumor vasculature were determined by immunostaining for blood and lymphatic vessel markers. Results: TLR4 expressed in tumor cells significantly increased rate of recurrence and metastasis, an event augmented by PXL treatment. Local inflammation was also enhanced by the PXL•TLR4 axis as illustrated by a 276-fold increase in IL-6 in lysates of TLR4+ tumors from treated mice as compared with samples of isogenic tumors with depleted TLR4. Activation of this pathway also increased cytokine levels in the blood and distant organs as indicted by 3-6 fold increase in IL-4 and IL-10 in LNs and lungs. These pro-inflammatory systemic changes promoted generation of myeloid progenitors in bone marrow and spleen evident by 4-5 fold increase in Ly6C+ cell population in TLR4-overexpressing tumors compared with those lacking TLR4. Importantly, activation of the PXL•TLR4 axis induced intratumoral formation of lymphatic vessels in HCC1806 tumors absent in all other experimental groups. Conclusion: These data imply that PXL therapy activates TLR4 often overexpressed in breast cancer. Activation of this pathway substantially increases local tumor and systemic inflammation leading to generation of myeloid progenitors that can promote metastasis by inducing angiogenesis and lymphatic vessel formation, particularly inside the tumor. These findings suggest that tumor expression of TLR4 may indicate poor prognosis and response to therapy, and that blocking the TLR4 pathway may improve current anti-cancer treatments. Citation Format: Sandeep Rajput, Lisa Volk-Draper, Kelly Hall, Sophia Ran. TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2014-771

Integrin α3β1 controls mRNA splicing that determines cyclooxygenase-2 (Cox-2) mRNA stability in breast cancer cells

It is unknown how cues from the tumor microenvironment can regulate post-transcriptional mechanisms, such as alternative splicing, that control genes that drive malignant growth. The induction of cyclooxygenase 2 (Cox-2) by integrin α3β1 in breast cancer cells can promote tumor progression. We have used RNAi to suppress α3β1 in human MDA-MB-231 breast cancer cells and then investigated changes in global gene expression. Numerous mRNAs, including Cox-2, show altered expression and/or alternative exon usage (AEU) in α3β1-deficient cells. AEU included patterns predicted to render an mRNA susceptible to degradation, such as 3'-UTR variations or retention of elements that target an mRNA for nonsense-mediated decay (NMD). PCR-based analysis of α3β1-deficient cells confirmed changes in Cox-2 mRNA that might target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 3'-UTR. Moreover, Cox-2 mRNA has reduced stability in α3β1-deficient cells, which is partially reversed by knockdown of the essential NMD factor UPF1. Our study identifies α3β1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that has potential for exploitation as a therapeutic target.

Abnormal expression of APRIL in colorectal cancer cells promotes tumor growth and metastasis

To investigate the effects of a proliferation-inducing ligand (APRlL) on colorectal cancer (CRC) cell growth and migration, and to observe the role of APRIL in CRC biological behavior. The siRNA plasmid vector targeting APRIL gene (APRIL-siRNA) was transfected into human colorectal cancer SW480 cells and recombinant human APRIL (rhAPRIL) was used to stimulate human colorectal cancer HCT-116 cells. Cell proliferation activity was analyzed using cell counting kit-8 (CCK-8), cell cycle was detected by flow cytometry, and the protein expression of cyclin D1, p21 and Bcl-2 was detected by Western blot analysis. Tumor cell migration and invasion were measured by Transwell chambers. RT-PCR was applied to examine the mRNA expression level of MMP-2 and MMP-9. APRIL-siRNA was used to transfect directly SW480 cells, which were injected subcutaneously into nude mice, then the tumor growth and metastasis were observed. Cell proliferation ability of APRIL-siRNA-transfected SW480 cells was drastically repressed, and the percentage of G0/G1 phase cells was significantly increased (t = 4.12, P < 0.05), accompanied with depressed cyclin D1, Bcl-2 expression and elevated p21 expression. Cell proliferation ability of rhAPRIL-stimulated HCT-116 cells was promoted with a decreased G0/G1 phase ratio (t = 3.31, P < 0.05). cyclin D1 and Bcl-2 protein expression was up-regulated while p21 was down-regulated by rhAPRIL stimulation. Metastatic and invasive capacities of APRIL-siRNA-transfected SW480 cells were significantly inhibited compared with their respective controls (both P < 0.05), accompanied with the deregulated MMP-2 and MMP-9 mRNA expression. Metastatic and invasive capacities of rhAPRIL-stimulated HCT-116 cells were promoted with up-regulated MMP-2 and MMP-9 mRNA expression(both P < 0.05). Tumor growth in the group transfected with APRIL-siRNA appeared to be slower than that in the control groups and the expression of MMP-2, MMP-9 in tumor tissues was depressed in the APRIL-siRNA group. APRIL facilitates tumor growth and metastasis, and is associated with carcinogenesis and prognosis. Our findings suggest that APRIL might be used as a novel target for the intervention and therapy of colorectal cancer.

Patent Highlights

Patent Highlights

Overexpression of Pregnancy-Associated Plasma Protein-A in Ovarian Cancer Cells Promotes Tumor Growth in Vivo

Pregnancy-associated plasma protein-A (PAPP-A) is an important regulatory component of the IGF system. Through proteolysis of inhibitory IGF binding proteins (IGFBPs), PAPP-A acts as a positive modulator of local IGF signaling in a variety of biological systems. A role of IGF in the progression of several common forms of human cancer is now emerging, and therapeutic intervention of IGF receptor signaling is currently being explored. However, little is known about the activities of other components of the IGF system in relation to cancer. We hypothesized that PAPP-A acts to enhance tumor growth in vivo. To test this hypothesis, we overexpressed wild-type PAPP-A or a mutant PAPP-A with markedly reduced IGFBP protease activity in SKOV3 cells, a human ovarian carcinoma cell line with low tumorigenic potential. In vitro, SKOV3 clones with elevated PAPP-A expression (PAPP-A-1, PAPP-A-28) showed accelerated anchorage-independent growth in soft agar assays compared to clones overexpressing mutant PAPP-A (E483Q-1, E483Q-5) and vector controls. PAPP-A-28, with the highest PAPP-A expression and IGFBP proteolytic activity, also had markedly increased cell invasion through Matrigel. In vivo, we found significantly accelerated tumor growth rates of PAPP-A-overexpressing SKOV3 clones compared with mutant PAPP-A and controls. Investigation of angiogenesis indicated that overexpression of PAPP-A favored development of mature tumor vasculature and that tumor precursors of PAPP-A-28 in particular had a significantly higher degree of vascularization months before obvious tumor development. In conclusion, our data show that PAPP-A proteolytic activity enhances the tumorigenic potential of ovarian cancer cells and establish a novel tumor growth-promoting role of PAPP-A.

Deregulation of the Pit-1 transcription factor in human breast cancer cells promotes tumor growth and metastasis

The Pit-1 transcription factor (also know as POU1F1) plays a critical role in cell differentiation during organogenesis of the anterior pituitary in mammals and is a transcriptional activator for pituitary gene transcription. Increased expression of Pit-1 has been reported in human tumorigenic breast cells. Here, we found that Pit-1 overexpression or knockdown in human breast cancer cell lines induced profound phenotypic changes in the expression of proteins involved in cell proliferation, apoptosis, and invasion. Some of these protumorigenic effects of Pit-1 were mediated by upregulation of Snai1, an inductor of the epithelial-mesenchymal transition. In immunodeficient mice, Pit-1 overexpression induced tumoral growth and promoted metastasis in lung. In patients with invasive ductal carcinoma of the breast and node-positive tumor, high expression of Pit-1 was significantly correlated with Snai1 positivity. Notably, in these patients elevated expression of Pit-1 was significantly and independently associated with the occurrence of distant metastasis. These findings suggest that Pit-1 could help to make a more accurate prognosis in patients with node-positive breast cancer and may represent a new therapeutic target.

Crosstalk between Mast Cells and Pancreatic Cancer Cells Contributes to Pancreatic Tumor Progression

To assess the clinical and pathologic significance of mast cell infiltration in human pancreatic cancer and evaluate crosstalk between mast cells and cancer cells in vitro. Immunohistochemistry for tryptase was done on 53 pancreatic cancer specimens. Mast cell counts were correlated with clinical variables and survival. Serum tryptase activity from patients with cancer was compared with patients with benign pancreatic disease. In vitro, the effect of pancreatic cancer-conditioned medium on mast cell migration was assessed. The effect of conditioned medium from the human mast cell line, LAD-2, on cancer and normal ductal cell proliferation was assessed by thymidine incorporation. Matrigel invasion assays were used to evaluate the effect of mast cell-conditioned medium on cancer cell invasion in the presence and absence of a matrix metalloproteinase inhibitor, GM6001. Mast cell infiltration was significantly increased in pancreatic cancer compared with normal pancreatic tissue (11.4 +/- 6.7 versus 2.0 +/- 1.4, P < 0.001). Increased infiltrating mast cells correlated with higher grade tumors (P < 0.0001) and worse survival. Patients with pancreatic cancer had elevated serum tryptase activity (P < 0.05). In vitro, AsPC1 and PANC-1 cells induced mast cell migration. Mast cell-conditioned medium induced pancreatic cancer cell migration, proliferation, and invasion but had no effect on normal ductal cells. Furthermore, the effect of mast cells on cancer cell invasion was, in large part, matrix metalloproteinase-dependent. Tumor-infiltrating mast cells are associated with worse prognosis in pancreatic cancer. In vitro, the interaction between mast cells and pancreatic cancer cells promotes tumor growth and invasion.

HYAL1-v1, An Alternatively Spliced Variant of HYAL1 Hyaluronidase: A Negative Regulator of Bladder Cancer

Tumor cells express HYAL1 hyaluronidase, which degrades hyaluronic acid. HYAL1 expression in bladder cancer cells promotes tumor growth, invasion, and angiogenesis. We previously described five alternatively spliced variants of HYAL1 that encode enzymatically inactive proteins. The HYAL1-v1 variant lacks a 30-amino acid sequence that is present in HYAL1. In this study, we examined whether HYAL1-v1 expression affects bladder cancer growth and invasion by stably transfecting HT1376 bladder cancer cells with a HYAL1-v1 cDNA construct. Although HYAL1-v1 transfectants expressed equivalent levels of enzymatically active HYAL1 protein when compared with vector transfectants, their conditioned medium had 4-fold less hyaluronidase activity due to a noncovalent complex formed between HYAL1 and HYAL1-v1 proteins. HYAL1-v1 transfectants grew 3- to 4-fold slower due to cell cycle arrest in the G(2)-M phase and increased apoptosis. In HYAL1-v1 transfectants, cyclin B1, cdc2/p34, and cdc25c levels were > or =2-fold lower than those in vector transfectants. The increased apoptosis in HYAL1-v1 transfectants was due to the extrinsic pathway involving Fas and Fas-associated death domain up-regulation, caspase-8 activation, and BID cleavage, leading to caspase-9 and caspase-3 activation and poly(ADP-ribose) polymerase cleavage. When implanted in athymic mice, HYAL1-v1-expressing tumors grew 3- to 4-fold slower and tumor weights at day 35 were 3- to 6-fold less than the vector tumors (P < 0.001). Whereas vector tumors were infiltrating and had high mitoses and microvessel density, HYAL1-v1 tumors were necrotic, infiltrated with neutrophils, and showed low mitoses and microvessel density. Therefore, HYAL-v1 expression may negatively regulate bladder tumor growth, infiltration, and angiogenesis.