Identifying and targeting key driver genes for collagen production within the 11q13/14 breast cancer amplicon.

Genetic studies indicate that breast cancer can be divided into several basic molecular groups. One of these groups, termed IntClust-2, is characterized by amplification of a small portion of chromosome 11 and has a median survival of only five years. Several cancer-relevant genes occupy this portion of chromosome 11, and it is thought that overexpression of a combination of driver genes in this region is responsible for the poor outcome of women in this group. In this study we used a gene editing method to knock out, one by one, each of 198 genes that are located within the amplified region of chromosome 11 and determined how much each of these genes contributed to the survival of breast cancer cells. In addition to well-known drivers such as CCND1 and PAK1 , we identified two different genes ( SERPINH1 and P4HA3 ), that encode proteins involved in collagen synthesis and organization. Using both in vitro and in vivo functional analyses, we determined that P4HA3 and/or SERPINH1 provide a critical driver function on IntClust-2 basic processes, such as viability, proliferation, and migration. Inhibiting these enzymes via genetic or pharmacologic means reduced collagen synthesis and impeded oncogenic signaling transduction in cell culture models, and a small-molecule inhibitor of P4HA3 was effective in treating 11q13 tumor growth in an animal model. As collagen has a well-known association with tissue stiffness and aggressive forms of breast cancer, we believe that the two genes we identified provide an opportunity for a new therapeutic strategy in IntClust-2 breast cancers.

GLCE rs3865014 (Val597Ile) polymorphism is associated with breast cancer susceptibility and triple-negative breast cancer in Siberian population.

d-Glucuronyl C5-epimerase (GLCE) is one of key enzymes in heparan sulfate biosynthesis and possesses tumour-suppressor function in breast carcinogenesis. Here, we investigated a potential involvement of GLCE polymorphism(s) in breast cancer development in Siberian women population. Comprehensive analysis of SNP databases revealed GLCE rs3865014 (Val597Ile) missense polymorphism as the main significantly present in human populations. According the TaqMan-based SNP assay, allele distributions for the rs3865014 (A>G) were similar in healthy Siberian women (n=136) and cancer patients (n=129) (A0,73:G0,27) and intermediate between the European and Asian populations, while genotype distributions were different, with the increase of AG rate in breast cancer patients (OR=1.76; 95% CI=1.04-1.90; P(Y)=0.035 χ2=4.44). Heterozygous AG genotype was associated with tumour size (OR=3.67, P(Y)=0.004), ER-negative tumours (OR=3.25, P(Y)=0.0028), triple-negative tumours (OR=4.94, P(Y)=0.015) but not menopausal status, PR and HER-2 status, local or distant metastasis. Homozygous GLCE genotypes (AA/GG) were more common for ER+PR+ luminal A breast cancer (OR=0.25, P(Y)=0.031). Loss-of-heterozigosity was identified in 5 of 51 breast tumours and the loss of G allele was associated with the decreased GLCE expression. Epidemiologic data for the GLCE SNP in different racial/ethnic groups demonstrated high AG genotype rates as a risk factor not for breast cancer incidence but for poor prognosis of the disease. The obtained data suggest an involvement of GLCE rs3865014 in breast cancer development. Heterozygous AG genotype might be a risk factor for breast cancer susceptibility in Siberian women and is associated with aggressive ER-negative and triple-negative cancer subtypes.

The Group I Pak inhibitor Frax-1036 sensitizes 11q13-amplified ovarian cancer cells to the cytotoxic effects of Rottlerin.

The p21-activated kinases (PAKs) are Cdc42/Rac-activated serine-threonine protein kinases that regulate several key cancer-relevant signaling pathways, such as the Mek/Erk, PI3K/Akt, and Wnt/ß-catenin cascades. Pak1 is frequently overexpressed and/or hyperactivated in different human cancers, including breast, ovary, prostate, and brain cancer. PAK1 genomic amplification at 11q13 is the most common mechanism of Pak1 hyperactivation, though Pak1 mRNA and/or protein may be overexpressed in the absence of gene amplification. In previous in vitro and in vivo studies we have shown that ovarian cancer cells with amplified/overexpressed Pak1 were significantly more sensitive to pharmacologic inhibition of Pak1 compared to cells without 11q13 amplification. In the present study we examined if additional signaling pathways might be targeted in tandem with the Group I Pak inhibitor Frax-1036 in ovarian cancer cells. Using the ICCB Known Bioactives Library, we found that the cytotoxic effect of Frax-1036 was significantly higher in combination with the PKCδ inhibitor, Rottlerin, suggesting that Pak inhibitors might be combined with other agents to treat 11q13-amplified ovarian cancer.

Reduced PAK1 activity sensitizes FA/BRCA-proficient breast cancer cells to PARP inhibition.

Cells that are deficient in homologous recombination, such as those that have mutations in any of the Fanconi Anemia (FA)/BRCA genes, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, FA/BRCA-deficient tumors represent a small fraction of breast cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. The gene encoding the serine-threonine protein kinase p21-activated kinase 1 (PAK1) is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme controls many cellular processes by phosphorylating both cytoplasmic and nuclear substrates. Here, we show that depletion or pharmacological inhibition of PAK1 down-regulated the expression of genes involved in the FA/BRCA pathway and compromised the ability of cells to repair DNA by Homologous Recombination (HR), promoting apoptosis and reducing colony formation. Combined inhibition of PAK1 and PARP in PAK1 overexpressing breast cancer cells had a synergistic effect, enhancing apoptosis, suppressing colony formation, and delaying tumor growth in a xenograft setting. Because reduced PAK1 activity impaired FA/BRCA function, inhibition of this kinase in PAK1 amplified and/or overexpressing breast cancer cells represents a plausible strategy for expanding the utility of PARP inhibitors to FA/BRCA-proficient cancers.

H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2 Heterodimerization.

The protein kinases Mst1 and Mst2 have tumor suppressor activity, but their mode of regulation is not well established. Mst1 and Mst2 are broadly expressed and may have certain overlapping functions in mammals, as deletions of both Mst1 and Mst2 together are required for tumorigenesis in mouse models [1-3]. These kinases act via a three-component signaling cascade comprising Mst1 and Mst2, the protein kinases Lats1 and Lats2, and the transcriptional coactivators Yap and Taz [4-6]. Mst1 and Mst2 contain C-terminal SARAH domains that mediate their homodimerization as well as heterodimerization with other SARAH domain-containing proteins, which may regulate Mst1/Mst2 activity. Here we show that, in addition to forming homodimers, Mst1 and Mst2 heterodimerize in cells, this interaction is mediated by their SARAH domains and is favored over homodimers, and these heterodimers have much-reduced protein kinase activity compared to Mst1 or Mst2 homodimers. Mst1/Mst2 heterodimerization is strongly promoted by oncogenic H-ras, and this effect requires activation of the Erk pathway. Cells lacking Mst1, in which Mst1/Mst2 heterodimers are not possible, are resistant to H-ras-mediated transformation and maintain active hippo pathway signaling compared to wild-type cells or cells lacking both Mst1 and Mst2. Our results suggest that H-ras, via an Erk-dependent mechanism, downregulates Mst1/Mst2 activity by inducing the formation of inactive Mst1/Mst2 heterodimers.

Tissue-specificity of heparan sulfate biosynthetic machinery in cancer.

Heparan sulfate (HS) proteoglycans are key components of cell microenvironment and fine structure of their polysaccharide HS chains plays an important role in cell-cell interactions, adhesion, migration and signaling. It is formed on non-template basis, so, structure and functional activity of HS biosynthetic machinery is crucial for correct HS biosynthesis and post-synthetic modification. To reveal cancer-related changes in transcriptional pattern of HS biosynthetic system, the expression of HS metabolism-involved genes (EXT1/2, NDST1/2, GLCE, 3OST1/HS3ST1, SULF1/2, HPSE) in human normal (fibroblasts, PNT2) and cancer (MCF7, LNCaP, PC3, DU145, H157, H647, A549, U2020, U87, HT116, KRC/Y) cell lines and breast, prostate, colon tumors was studied. Real-time RT-PCR and Western-blot analyses revealed specific transcriptional patterns and expression levels of HS biosynthetic system both in different cell lines in vitro and cancers in vivo. Balance between transcriptional activities of elongation- and post-synthetic modification- involved genes was suggested as most informative parameter for HS biosynthetic machinery characterization. Normal human fibroblasts showed elongation-oriented HS biosynthesis, while PNT2 prostate epithelial cells had modification-oriented one. However, cancer epithelial cells demonstrated common tendency to acquire fibroblast-like elongation-oriented mode of HS biosynthetic system. Surprisingly, aggressive metastatic cancer cells (U2020, DU145, KRC/Y) retained modification-oriented HS biosynthesis similar to normal PNT2 cells, possibly enabling the cells to keep like-to-normal cell surface glycosylation pattern to escape antimetastatic control. The obtained results show the cell type-specific changes of HS-biosynthetic machinery in cancer cells in vitro and tissue-specific changes in different cancers in vivo, supporting a close involvement of HS biosynthetic system in carcinogenesis.

Pak2 restrains endomitosis during megakaryopoiesis and alters cytoskeleton organization.

Megakaryocyte maturation and polyploidization are critical for platelet production; abnormalities in these processes are associated with myeloproliferative disorders, including thrombocytopenia. Megakaryocyte maturation signals through cascades that involve p21-activated kinase (Pak) function; however, the specific role for Pak kinases in megakaryocyte biology remains elusive. Here, we identify Pak2 as an essential effector of megakaryocyte maturation, polyploidization, and proplatelet formation. Genetic deletion of Pak2 in murine bone marrow is associated with macrothrombocytopenia, altered megakaryocyte ultrastructure, increased bone marrow megakaryocyte precursors, and an elevation of mature CD41(+) megakaryocytes, as well as an increased number of polyploid cells. In Pak2(-/-) mice, platelet clearance rate was increased, as was production of newly synthesized, reticulated platelets. In vitro, Pak2(-/-) megakaryocytes demonstrate increased polyploidization associated with alterations in ß1-tubulin expression and organization, decreased proplatelet extensions, and reduced phosphorylation of the endomitosis regulators LIM domain kinase 1, cofilin, and Aurora A/B/C. Together, these data establish a novel role for Pak2 as an important regulator of megakaryopoiesis, polyploidization, and cytoskeletal dynamics in developing megakaryocytes.

Molecular pathways: targeting the kinase effectors of RHO-family GTPases.

RHO GTPases, members of the RAS superfamily of small GTPases, are adhesion and growth factor-activated molecular switches that play important roles in tumor development and progression. When activated, RHO-family GTPases such as RAC1, CDC42, and RHOA, transmit signals by recruiting a variety of effector proteins, including the protein kinases PAK, ACK, MLK, MRCK, and ROCK. Genetically induced loss of RHO function impedes transformation by a number of oncogenic stimuli, leading to an interest in developing small-molecule inhibitors that either target RHO GTPases directly, or that target their downstream protein kinase effectors. Although inhibitors of RHO GTPases and their downstream signaling kinases have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to facilitate pharmaceutical research and development and is a promising therapeutic strategy.

D-glucuronyl C5-epimerase cell type specifically affects angiogenesis pathway in different prostate cancer cells.

D-glucuronyl C5-epimerase (GLCE) is involved in breast and lung carcinogenesis as a potential tumor suppressor gene, acting through inhibition of tumor angiogenesis and invasion/metastasis pathways. However, in prostate tumors, increased GLCE expression is associated with advanced disease, suggesting versatile effects of GLCE in different cancers. To investigate further the potential cancer-promoting effect of GLCE in prostate cancer, GLCE was ectopically re-expressed in morphologically different LNCaP and PC3 prostate cancer cells. Transcriptional profiles of normal PNT2 prostate cells, LNCaP, PC3 and DU145 prostate cancer cells, and GLCE-expressing LNCaP and PC3 cells were determined. Comparative analysis revealed the genes whose expression was changed in prostate cancer cells compared with normal PNT2 cells, and those differently expressed between the cancer cell lines (ACTA2, IL6, SERPINE1, TAGLN, SEMA3A, and CDH2). GLCE re-expression influenced mainly angiogenesis-involved genes (ANGPT1, SERPINE1, IGF1, PDGFB, TNF, IL8, TEK, IFNA1, and IFNB1) but in a cell type-specific manner (from basic deregulation of angiogenesis in LNCaP cells to significant activation in PC3 cells). Invasion/metastasis pathway was also affected (MMP1, MMP2, MMP9, S100A4, ITGA1, ITGB3, ERBB2, and FAS). The obtained results suggest activation of angiogenesis as a main molecular mechanism of pro-oncogenic effect of GLCE in prostate cancer. GLCE up-regulation plus expression pattern of a panel of six genes, discriminating morphologically different prostate cancer cell sub-types, is suggested as a potential marker of aggressive prostate cancer.

Heterogeneity of d-glucuronyl C5-epimerase expression and epigenetic regulation in prostate cancer.

Heparansulfate proteoglycans (HSPG) play an important role in cell-cell and cell-matrix interactions and signaling, and one of the key enzymes in heparansulfate biosynthesis is d-glucuronyl C5-epimerase (GLCE). A tumor suppressor function has been demonstrated for GLCE in breast and lung carcinogenesis; however, no data are available as to the expression and regulation of the gene in prostate cancer. In this study, decreased GLCE expression was observed in 10% of benign prostate hyperplasia (BPH) tissues and 53% of prostate tumors, and increased GLCE mRNA levels were detected in 49% of BPH tissues and 21% of tumors. Statistical analysis showed a positive correlation between increased GLCE expression and Gleason score, TNM staging, and prostate-specific antigen (PSA) level in the prostate tumors (Pearson correlation coefficients GLCE/Gleason = 0.56, P < 0.05; GLCE/TNM = 0.62, P < 0.05; and GLCE/PSA = 0.88, P < 0.01), suggesting GLCE as a candidate molecular marker for advanced prostate cancer. Immunohistochemical analysis revealed an intratumoral heterogeneity of GLCE protein levels both in BPH and prostate cancer cells, resulting in a mixed population of GLCE-expressing and nonexpressing epithelial cells in vivo. A model experiment on normal (PNT2) and prostate cancer (LNCaP, PC3, DU145) cell lines in vitro showed a 1.5- to 2.5-fold difference in GLCE expression levels between the cancer cell lines and an overall decrease in GLCE expression in cancer cells. Methyl-specific polymerase chain reaction (PCR), bisulfite sequencing, and deoxy-azacytidin (aza-dC) treatment identified differential GLCE promoter methylation (LNCaP 70-72%, PC3 32-35%, DU145, and PNT2 no methylation), which seems to contribute to heterogeneous GLCE expression in prostate tumors. The obtained results reveal the complex deregulation of GLCE expression in prostatic diseases compared with normal prostate tissue and suggest that GLCE may be used as a potential model to study the functional role of intratumor cell heterogeneity in prostate cancer progression.

miRNA-218 contributes to the regulation of D-glucuronyl C5-epimerase expression in normal and tumor breast tissues.

microRNAs (miRNAs) are key posttranscriptional regulators of gene expression. In the present study, regulation of tumor-suppressor gene D-glucuronyl C5-epimerase (GLCE) by miRNA-218 was investigated. Significant downregulation of miRNA-218 expression was shown in primary breast tumors. Exogenous miRNA-218/anti-miRNA-218 did not affect GLCE mRNA but regulated GLCE protein level in MCF7 breast carcinoma cells in vitro. Comparative analysis showed a positive correlation between miRNA-218 and GLCE mRNA, and negative correlation between miRNA-218 and GLCE protein levels in breast tissues and primary tumors in vivo, supporting a direct involvement of miRNA-218 in posttranscriptional regulation of GLCE in human breast tissue. A common scheme for the regulation of GLCE expression in normal and tumor breast tissues is suggested.

The TCF4/ß-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer.

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/ß-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/ß-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/ß-catenin mRNA levels and activated TCF4/ß-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/ß-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and ß-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/ß-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/ß-catenin complex activity.

Integrin alpha9 (ITGA9) expression and epigenetic silencing in human breast tumors.

Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in normal human breast tissue, primary breast tumors and breast cancer cell line MCF7 were studied. It was shown that integrin alpha9 is expressed in normal human breast tissue. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (possible aberrations in the ITGA9 mRNA structure were supposed in 11% of tumors). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5'-untranslated region of ITGA9 was observed. 5-aza-dC treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-dC/Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer with a possible involvement of other yet unidentified molecular pathways.

Antiproliferative effect of D-glucuronyl C5-epimerase in human breast cancer cells.

BACKGROUND: D-glucuronyl C5-epimerase (GLCE) is one of the key enzymes in the biosynthesis of heparansulfate proteoglycans. Down-regulation of GLCE expression in human breast tumours suggests a possible involvement of the gene in carcinogenesis. In this study, an effect of GLCE ectopic expression on cell proliferation and viability of breast carcinoma cells MCF7 in vitro and its potential molecular mechanisms were investigated. RESULTS: D-glucuronyl C5-epimerase expression was significantly decreased in MCF7 cells compared to normal human breast tissue. Re-expression of GLCE inhibited proliferative activity of MCF7 cells according to CyQUANT NF Cell Proliferation Assay, while it did not affect their viability in Colony Formation Test. According to Cancer PathFinder RT Profiler PCR Array, antiproliferative effect of GLCE in vitro could be related to the enhanced expression of tumour suppressor genes р53 (+3.3 fold), E2F1 (+3.00 fold), BRCA1 (+3.5 fold), SYK (+8.1 fold) and apoptosis-related genes BCL2 (+4.2 fold) and NFKB1 (+2.6 fold). Also, GLCE re-expression in MCF7 cells considerably changed the expression of some genes involved in angiogenesis (IL8, +4.6 fold; IFNB1, +3.9 fold; TNF, +4.6 fold and TGFB1, -5.7 fold) and invasion/metastasis (SYK, +8.1 fold; NME1, +3.96 fold; S100A4, -4.6 fold). CONCLUSIONS: The ability of D-glucuronyl С5-epimerase to suppress proliferation of breast cancer cells MCF7 through the attenuated expression of different key genes involved in cell cycle regulation, angiogenesis and metastasis molecular pathways supports the idea on the involvement of the gene in regulation of breast cancer cell proliferation.