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TGF-² Signaling Pathway in Lung Adenocarcinoma Invasion

      Abstract

      The histologic distinction between bronchioloalveolar carcinoma and other adenocarcinomas is tissue invasion. The clinical importance of lung adenocarcinoma invasion is supported by several recent studies indicating that the risk of death in nonmucinous bronchioloalveolar carcinoma is significantly lower than that of pure invasive tumors and in tumors with greater than 0.5 cm of fibrosis or linear invasion. Using microarray gene expression profiling of human tumors, dysregulation of transforming growth factor-² signaling was identified as an important mediator of tumor invasion. Subsequent studies showed that the CC chemokine regulated on activation, normal T cell expressed, and presumably secreted was up-regulated in invasive tumors and was required for invasion in cells with repressed levels of the transforming growth factor-² type II receptor. Taken together, these studies illustrate how information gained from global expression profiling of tumors can be used to identify key pathways and genes mediating tumor growth, invasion, and metastasis.

      Keywords

      The World Health Organization subclassifies adenocarcinoma (AC) based on predominant cell morphology and growth pattern.
      • Brambilla E
      • Travis WD
      • Colby TV
      • Corrin B
      • Shimosato Y
      The new World Health Organization classification of lung tumours.
      The histologic distinction between bronchioloalveolar carcinoma (BAC) and other ACs is tissue invasion. BAC tumor cells are cuboidal to columnar, with or without mucin, that grow in a noninvasive fashion along alveolar walls. Invasion, defined as tumor disruption of the alveolar basement membrane, is present in other subtypes of AC. ACs with mixed subtypes frequently contain regions of lepidic/noninvasive tumor at the periphery of invasive tumor.
      Recent clinical reports suggest that the prognosis and radiographic appearance of BAC is unique and may support modifying the clinical approach to lung ACs according to histologic subtype. Metastases to lymph nodes and extrathoracic organs are unusual in nonmucinous BAC. The mean 5-year survival for stage I BAC and other ACs is 81 and 55%, respectively.
      • Rena O
      • Papalia E
      • Ruffini E
      • et al.
      Stage I pure bronchioloalveolar carcinoma: recurrences, survival and comparison with adenocarcinoma of the lung.
      Recent reports suggest that for stage IA BAC, limited resections rather than lobectomy, which is the current standard resection for stage IA AC, may be curative.
      • Nakayama H
      • Yamada K
      • Saito H
      • et al.
      Sublobar resection for patients with peripheral small adenocarcinomas of the lung: surgical outcome is associated with features on computed tomographic imaging.
      Notably, low-dose chest computed tomographic screening detected lung cancer is more likely to be AC than conventionally detected cancer (75% versus 40%).
      • Henschke CI
      • McCauley DI
      • Yankelevitz DF
      • et al.
      Early Lung Cancer Action Project: overall design and findings from baseline screening.
      • Swensen SJ
      • Jett JR
      • Sloan JA
      • et al.
      Screening for lung cancer with low-dose spiral computed tomography.
      In addition, 25% of screen detected cancers are BAC. As a result, the identification of invasion in screen-detected malignancy may in the future guide a therapeutic decision of limited versus anatomic resection.
      Paralleling malignancies in other organs, such as breast and cervix, where tumors are defined as noninvasive (in situ carcinoma), microinvasive (microscopic invasion), or as invasive carcinomas, the extent of the invasive component seen in lung AC is associated with clinical outcomes. The clinical importance of lung AC invasion is supported by several recent studies,
      • Rena O
      • Papalia E
      • Ruffini E
      • et al.
      Stage I pure bronchioloalveolar carcinoma: recurrences, survival and comparison with adenocarcinoma of the lung.
      • Maeshima AM
      • Niki T
      • Maeshima A
      • Yamada T
      • Kondo H
      • Matsuno Y
      Modified scar grade: a prognostic indicator in small peripheral lung adenocarcinoma.
      • Sakurai H
      • Maeshima A
      • Watanabe S
      • et al.
      Grade of stromal invasion in small adenocarcinoma of the lung: histopathological minimal invasion and prognosis.
      • Suzuki K
      • Yokose T
      • Yoshida J
      • et al.
      Prognostic significance of the size of central fibrosis in peripheral adenocarcinoma of the lung.
      • Yim J
      • Zhu LC
      • Chiriboga L
      • Watson HN
      • Goldberg JD
      • Moreira AL
      Histologic features are important prognostic indicators in early stages lung adenocarcinomas.
      indicating that the risk of death in nonmucinous BAC is significantly lower than that of pure invasive tumors and in tumors with greater than 0.5 cm of fibrosis or linear invasion. In 200 cases of small ACs (diameter <3 cm), Yokose et al.
      • Yokose T
      • Suzuki K
      • Nagai K
      • Nishiwaki Y
      • Sasaki S
      • Ochiai A
      Favorable and unfavorable morphological prognostic factors in peripheral adenocarcinoma of the lung 3 cm or less in diameter.
      reported no deaths among 66 BAC cases. In 484 cases of BAC and AC, Terasaki et al.
      • Terasaki H
      • Niki T
      • Matsuno Y
      • et al.
      Lung adenocarcinoma with mixed bronchioloalveolar and invasive components: clinicopathological features, subclassification by extent of invasive foci, and immunohistochemical characterization.
      reported that lymph node involvement was absent in all BAC and was present in 20% of ACs that had an invasive area greater than 5 mm. Similarly, among 178 patients with resected lung AC, we found 5-year survival rates of 100 and 90% for patients with BAC or tumors with invasive length less than 6 mm, respectively.
      • Borczuk AC
      • Qian F
      • Kazeros A
      • et al.
      Invasive size is an independent predictor of survival in pulmonary adenocarcinoma.
      Together, these studies suggest that noninvasive tumors are biologically indolent and that invasion increases the risk of metastatic disease and death in solitary mixed subtype tumors.
      Invasion is the first step of carcinoma metastasis, in which epithelial cells lose cell-cell adhesion, gain motility, and invade into adjacent stroma. Subsequent steps of metastasis include vascular intravasation and extravasation, establishment of a metastatic niche, and angiogenesis.
      • Fidler IJ
      The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited.
      Tumor invasiveness, the morphologic characteristic that distinguishes BAC from AC, is determined by the interaction of tumor cells with the surrounding stroma.
      • Bissell MJ
      • Radisky D
      Putting tumours in context.
      • Elenbaas B
      • Weinberg RA
      Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation.
      We
      • Borczuk AC
      • Kim HK
      • Yegen HA
      • Friedman RA
      • Powell CA
      Lung adenocarcinoma global profiling identifies type II transforming growth factor-beta receptor as a repressor of invasiveness.
      and others
      • Takeuchi T
      • Tomida S
      • Yatabe Y
      • et al.
      Expression profile-defined classification of lung adenocarcinoma shows close relationship with underlying major genetic changes and clinicopathologic behaviors.
      • Motoi N
      • Szoke J
      • Riely GJ
      • et al.
      Lung adenocarcinoma: modification of the 2004 WHO mixed subtype to include the major histologic subtype suggests correlations between papillary and micropapillary adenocarcinoma subtypes, EGFR mutations and gene expression analysis.
      • Beer DG
      • Kardia SL
      • Huang CC
      • et al.
      Gene-expression profiles predict survival of patients with lung adenocarcinoma.
      • Director's Challenge Consortium for the Molecular Classification of Lung Adenocarcinoma
      • Shedden K
      • Taylor JM
      • Enkemann SA
      • et al.
      Gene expression-based survival prediction in lung adenocarcinoma: a multi-site, blinded validation study.
      have used microarray gene expression profiling of lung AC to identify signatures associated with histology and invasion. The results of unsupervised analyses, in which the specimens are sorted into groups in a dendogram based on similarity of gene expression, show lung ACs segregate into three major branches comprised predominantly of BAC, AC-mixed subtype, and pure invasive tumors. These results provide biologic plausibility to support the notion that these AC subtypes are distinct entities. Taken together with the clinical prognostic data, these studies have motivated efforts to reinforce the designation of purely noninvasive tumors and to create a designation for minimally invasive tumors in a revision of the World Health Organistaion lung AC classification scheme.
      To identify molecular pathways important for mediating the acquisition of invasion by lung AC, we performed supervised analysis of mRNA microarray data to identify genes differentially expressed in noninvasive BAC and in AC-mixed type tumors. Among the genes differentially expressed in the progression from BAC to invasive tumors was the transforming growth factor-² (TGF-²) type II receptor (T²RII), which was less highly expressed by AC-mixed and solid invasive tumors compared with BAC. This finding, which suggested that T²RII repression was required for lung AC invasion, is supported by genetic models combining targeted deletion of T²RII with other oncogenic events such as adenomatosis polyposis coli mutation in colon tumors and KRAS mutations in pancreatic and oropharyngeal carcinomas.
      • Ijichi H
      • Chytil A
      • Gorska AE
      • et al.
      Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression.
      • Lu SL
      • Herrington H
      • Reh D
      • et al.
      Loss of transforming growth factor-beta type II receptor promotes metastatic head-and-neck squamous cell carcinoma.
      • Munoz NM
      • Upton M
      • Rojas A
      • et al.
      Transforming growth factor beta receptor type II inactivation induces the malignant transformation of intestinal neoplasms initiated by Apc mutation.
      The phenotypes of these TGF-² receptor cancer models clearly demonstrate the importance of TGF-² signaling in tumor invasion.
      TGF-², the ligand for the TGF-² type II receptor is a pleiotropic cytokine comprised of family members TGF-² 1, 2, and 3 that regulate tissue homeostasis and prevent tumor initiation by inhibiting cellular proliferation, differentiation, and survival.
      • Siegel PM
      • Massague J
      Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer.
      It is secreted as a latent molecule and is activated by cleavage by proteases and other molecules.
      • Yoshinaga K
      • Obata H
      • Jurukovski V
      • et al.
      Perturbation of transforming growth factor (TGF)-beta1 association with latent TGF-beta binding protein yields inflammation and tumors.
      Signaling primarily occurs through SMAD protein dependent pathways whereby ligand binding to TBRII induces phosphorylation and activation of TGF-² type I receptor (T²RI). After interaction with T²RI, phosphorylated SMAD2 and SMAD3 dissociate to form a heterotrimeric complex with SMAD4 and translocate into the nucleus to regulate gene transcription (Fig. 1A). TGF-² signaling may also proceed via less well-understood SMAD independent pathways (Fig. 1B). These “noncanonical” pathways involve various signaling cascades including Ras/ERK, Rho/ROCK, and TAK1/MAPK and are likely to have important roles in mediating the protumorigenic effects of TGF-².
      • Massague J
      How cells read TGF-beta signals.
      Depending on context, TGF-² signaling may alternatively function to suppress tumor growth or to promote tumor cell invasion and metastasis.
      • Derynck R
      • Akhurst RJ
      • Balmain A
      TGF-beta signaling in tumor suppression and cancer progression.
      • Elliott RL
      • Blobe GC
      Role of transforming growth factor beta in human cancer.
      • Tang B
      • Vu M
      • Booker T
      • et al.
      TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression.
      • Roberts AB
      • Wakefield LM
      The two faces of transforming growth factor beta in carcinogenesis.
      Figure thumbnail gr1
      FIGURE 1TGF-² signaling occurs primarily via SMAD dependent pathways. A, ligand binding to the TGF-² type II receptor (T²RII) induces phosphorylation and activation of type I receptor (T²RI), which phosphorylates and activates the receptor complex SMAD2 and SMAD3. Dissociated SMAD2/3 forms a heterotrimeric complex with SMAD4 that translocates into the nucleus to regulate gene transcription. B, TGF-² signaling may also proceed via SMAD-independent pathways that involve various signaling cascades including Ras/ERK, Rho/ROCK, and TAK1/MAPK. These noncanonical pathways are likely to have important roles in mediating the protumorigenic effects of TGF-². C, The histologic distinction between bronchioloalveolar carcinoma (BAC) and other adenocarcinomas is tissue invasion. Invasion requires loss of cell-cell adhesion, migration, membrane degradation with vascular intravasation and extravasation, establishment of the metastatic niche angiogenesis and recruitment of stromal elements (top panel). We have shown that repression of TGF-² type II receptor in lung adenocarcinoma cells increases invasiveness and have used microarray analyses and inhibitor studies to identify the CC chemokine RANTES as an important mediator of lung adenocarcinoma invasion in T²RII-deficient tumors. Reprinted with permission from Borczuk AC, Toonkel RL, Powell CA. Genomics of lung cancer. Proceedings of the American Thoracic Society 2009;6:152–158. Official Journal of the American Thoracic Society. © American Thoracic Society.
      • Borczuk AC
      • Toonkel RL
      • Powell CA
      Genomics of lung cancer.
      .

      TGF-² as a Tumor Suppressor

      Although recent research has focused primarily on TGF-² receptor alterations, tumors may use various mechanisms anywhere along the signaling cascade to circumvent the inhibitory effects of TGF-².
      • Markowitz S
      • Wang J
      • Myeroff L
      • et al.
      Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability.
      • Kang SH
      • Bang YJ
      • Im YH
      • et al.
      Transcriptional repression of the transforming growth factor-beta type I receptor gene by DNA methylation results in the development of TGF-beta resistance in human gastric cancer.
      • Bierie B
      • Moses HL
      TGF-beta and cancer.
      • Levy L
      • Hill CS
      Alterations in components of the TGF-beta superfamily signaling pathways in human cancer.
      • Kim SJ
      • Im YH
      • Markowitz SD
      • Bang YJ
      Molecular mechanisms of inactivation of TGF-beta receptors during carcinogenesis.
      Type II receptor genetic alterations are well characterized in gastrointestinal tumors in which 25% of colorectal carcinomas have missense mutations associated with microsatellite instability. Animals with targeted deletion of T²RII in the colonic epithelium demonstrate increased tumor progression from adenomas to invasive carcinomas
      • Biswas S
      • Chytil A
      • Washington K
      • et al.
      Transforming growth factor beta receptor type II inactivation promotes the establishment and progression of colon cancer.
      similar to human colorectal tumors with loss of type II receptor.
      • Grady WM
      • Rajput A
      • Myeroff L
      • et al.
      Mutation of the type II transforming growth factor-beta receptor is coincident with the transformation of human colon adenomas to malignant carcinomas.
      In breast carcinoma models, mammary tumors in animals with targeted deletion of T²RII demonstrated increased progression and metastases.
      • Forrester E
      • Chytil A
      • Bierie B
      • et al.
      Effect of conditional knockout of the type II TGF-beta receptor gene in mammary epithelia on mammary gland development and polyomavirus middle T antigen induced tumor formation and metastasis.
      A recent case-control study in human breast tumors indicated that within breast hyperplasia specimens, the proportion of cells with decreased type II receptor immunostaining was associated with increased risk for the development of invasive breast cancer.
      • Gobbi H
      • Dupont WD
      • Simpson JF
      • et al.
      Transforming growth factor-beta and breast cancer risk in women with mammary epithelial hyperplasia.
      Multiple lung cancer cell lines, both small cell
      • Hougaard S
      • Norgaard P
      • Abrahamsen N
      • Moses HL
      • Spang-Thomsen M
      • Skovgaard Poulsen H
      Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines.
      • Damstrup L
      • Rygaard K
      • Spang-Thomsen M
      • Skovgaard Poulsen H
      Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines.
      • de Jonge RR
      • Garrigue-Antar L
      • Vellucci VF
      • Reiss M
      Frequent inactivation of the transforming growth factor beta type II receptor in small-cell lung carcinoma cells.
      and non-small cell,
      • Kim WS
      • Park C
      • Hong SK
      • Park BK
      • Kim HS
      • Park K
      Microsatellite instability (MSI) in non-small cell lung cancer (NSCLC) is highly associated with transforming growth factor-beta type II receptor (TGF-beta RII) frameshift mutation.
      • Kim TK
      • Mo EK
      • Yoo CG
      • et al.
      Alteration of cell growth and morphology by overexpression of transforming growth factor beta type II receptor in human lung adenocarcinoma cells.
      • Park C
      • Kim WS
      • Choi Y
      • Kim H
      • Park K
      Effects of transforming growth factor beta (TGF-beta) receptor on lung carcinogenesis.
      • Anumanthan G
      • Halder SK
      • Osada H
      • et al.
      Restoration of TGF-beta signalling reduces tumorigenicity in human lung cancer cells.
      demonstrate reduced expression TGF²RII. This repression is accompanied by marked reductions in TGF-² mediated growth suppression which is rescued after restoration of the receptor. In human lung tumor specimens, type II receptor repression is evident in approximately 40% of lung ACs overall and in up to 100% of poorly differentiated ACs.
      • Kang Y
      • Prentice MA
      • Mariano JM
      • et al.
      Transforming growth factor-beta 1 and its receptors in human lung cancer and mouse lung carcinogenesis.
      Mechanisms of repression include epigenetic silencing,
      • Zhang HT
      • Chen XF
      • Wang MH
      • et al.
      Defective expression of transforming growth factor beta receptor type II is associated with CpG methylated promoter in primary non-small cell lung cancer.
      microsatellite instability, and frameshift mutations involving the poly(A) tract.
      • Kim WS
      • Park C
      • Hong SK
      • Park BK
      • Kim HS
      • Park K
      Microsatellite instability (MSI) in non-small cell lung cancer (NSCLC) is highly associated with transforming growth factor-beta type II receptor (TGF-beta RII) frameshift mutation.
      For the TGF-² type I receptor, mRNA repression is detectable in non-small cell lung cancer,
      • Zhao J
      • Liu Z
      • Li W
      • Liu X
      • Chen XF
      • Zhang HT
      Infrequently methylated event at sites −362 to −142 in the promoter of TGF beta R1 gene in non-small cell lung cancer.
      and recent studies indicate that T²RI SNP variants are associated with an increased risk of lung cancer.
      • Zhang HT
      • Fei QY
      • Chen F
      • et al.
      Mutational analysis of the transforming growth factor beta receptor type I gene in primary non-small cell lung cancer.
      • Kang HG
      • Chae MH
      • Park JM
      • et al.
      Polymorphisms in TGF-beta1 gene and the risk of lung cancer.
      • Park KH
      • Lo Han SG
      • Whang YM
      • et al.
      Single nucleotide polymorphisms of the TGFB1 gene and lung cancer risk in a Korean population.

      TGF-² as a Tumor Promoter

      Several tumors, including those arising in the lung
      • Barthelemy-Brichant N
      • David JL
      • Bosquee L
      • et al.
      Increased TGFbeta1 plasma level in patients with lung cancer: potential mechanisms.
      • Lee JC
      • Lee KM
      • Kim DW
      • Heo DS
      Elevated TGF-beta1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients.
      • Domagala-Kulawik J
      • Hoser G
      • Safianowska A
      • Grubek-Jaworska H
      • Chazan R
      Elevated TGF-beta1 concentration in bronchoalveolar lavage fluid from patients with primary lung cancer.
      express high levels of the TGF-², which correlates with tumor progression and clinical prognosis.
      • Levy L
      • Hill CS
      Alterations in components of the TGF-beta superfamily signaling pathways in human cancer.
      • Bruna A
      • Darken RS
      • Rojo F
      • et al.
      High TGFbeta-Smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene.
      • Hasegawa Y
      • Takanashi S
      • Kanehira Y
      • Tsushima T
      • Imai T
      • Okumura K
      Transforming growth factor-beta1 level correlates with angiogenesis, tumor progression, and prognosis in patients with nonsmall cell lung carcinoma.
      • Saito H
      • Tsujitani S
      • Oka S
      • et al.
      The expression of transforming growth factor-beta1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma.
      • Tsushima H
      • Kawata S
      • Tamura S
      • et al.
      High levels of transforming growth factor beta 1 in patients with colorectal cancer: association with disease progression.
      • Wikstrom P
      • Stattin P
      • Franck-Lissbrant I
      • Damber JE
      • Bergh A
      Transforming growth factor beta1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer.
      TGF-² signaling promotes epithelial to mesenchymal transition, a characteristic of invasive and metastatic cells,
      • Oft M
      • Heider KH
      • Beug H
      TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis.
      • Deckers M
      • van Dinther M
      • Buijs J
      • et al.
      The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells.
      with constitutive activation of TGF-² or T²RI leading to increased metastases in animal models of breast cancer.
      • Muraoka-Cook RS
      • Kurokawa H
      • Koh Y
      • et al.
      Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors.
      • Muraoka-Cook RS
      • Shin I
      • Yi JY
      • et al.
      Activated type I TGFbeta receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression.
      • Siegel PM
      • Shu W
      • Cardiff RD
      • Muller WJ
      • Massague J
      Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis.
      Similarly, blockade of TGF-² signaling via either dominant negative expression of SMAD3 or defective T²RI leads to decreased lung metastases.
      • Tian F
      • Byfield SD
      • Parks WT
      • et al.
      Smad-binding defective mutant of transforming growth factor beta type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.
      • Tian F
      • DaCosta Byfield S
      • Parks WT
      • et al.
      Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.
      Systemic inhibition of TGF-² has been shown to suppress metastasis
      • Bandyopadhyay A
      • Agyin JK
      • Wang L
      • et al.
      Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor.
      • Yang YA
      • Dukhanina O
      • Tang B
      • et al.
      Lifetime exposure to a soluble TGF-beta antagonist protects mice against metastasis without adverse side effects.
      • Biswas S
      • Guix M
      • Rinehart C
      • et al.
      Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression.
      • Nam JS
      • Terabe M
      • Mamura M
      • et al.
      An anti-transforming growth factor beta antibody suppresses metastasis via cooperative effects on multiple cell compartments.
      and TGF-² overexpression by non-small cell lung cancer specimens was found by multivariate analysis to be an independent risk factor for pulmonary metastasis.
      • Saji H
      • Nakamura H
      • Awut I
      • et al.
      Significance of expression of TGF-beta in pulmonary metastasis in non-small cell lung cancer tissues.
      How do we reconcile these findings with those suggesting TGF-² is a tumor suppressor?
      Context dependency in terms of cell type, tumor stage, and mode of inhibition of TGF-² signaling are important. Other important issues are the degree of repression of TGF-² receptor levels and the stromal response to TGF-² signaling inhibition. Rojas et al.
      • Rojas A
      • Padidam M
      • Cress D
      • Grady WM
      TGF-beta receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-beta.
      have shown that different levels of repression of the TGF-² receptor are associated with differences in the activation of the SMAD and MAPK pathways such that at lower levels of TGF-² receptor activation, the protumorigenic non-SMAD signaling pathways dominate. Yang et al. showed that targeted deletion of T²RII in the mammary epithelium promoted breast cancer metastases through the CXCL5/CXCR2 chemokine axis mediated recruitment of Gr-1+/CD11b+ myeloid derived suppressor cells. Increased stromal TGF-² levels at the invasive front of tumors was shown to be important for tumor progression and for inhibition of tumor immunosurveillance.
      • Yang L
      • Huang J
      • Ren X
      • et al.
      Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis.

      Chemokine Signaling in Human Tumors with Repressed T²RII Expression—CCL5

      Our results in lung AC and in in vitro systems indicate that repression of the TGF-² type II receptor increases invasiveness. We have shown that activation of SMAD2 and Akt are lower in T²RII knockdown cells, whereas p38 activation is slightly increased.
      • Borczuk AC
      • Kim HK
      • Yegen HA
      • Friedman RA
      • Powell CA
      Lung adenocarcinoma global profiling identifies type II transforming growth factor-beta receptor as a repressor of invasiveness.
      We expect that TGF-² signaling in cells with moderately reduced type II receptor levels persists in the invasive tumors and in the knockdown cells and that SMAD independent pathways modulate this effect.
      • Rojas A
      • Padidam M
      • Cress D
      • Grady WM
      TGF-beta receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-beta.
      • Bhowmick NA
      • Zent R
      • Ghiassi M
      • McDonnell M
      • Moses HL
      Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38mapk and epithelial plasticity.
      We used a tumor cell invasion system and microarray analysis to identify and characterize downstream mediators of TGF-² signaling important for lung AC invasion.
      • Borczuk AC
      • Kim HK
      • Yegen HA
      • Friedman RA
      • Powell CA
      Lung adenocarcinoma global profiling identifies type II transforming growth factor-beta receptor as a repressor of invasiveness.
      Among potential mediators identified was the CC (or ²-chemokine) family member CCL5 (regulated on activation, normal T cell expressed, and presumably secreted [RANTES]), which was up-regulated by invasive tumors and T²RII knockdown cells. RANTES is involved in immunoregulatory and inflammatory processes and is secreted by T cells and other inflammatory cells, stromal cells, as well as tumor cells and normal bronchial epithelium. RANTES is a ligand for chemokine receptors CCR1, CCR3, CCR4, and CCR5, which are expressed on epithelial cells, macrophages, lymphocytes, dendritic cells, and stromal cells.
      • Fraziano M
      • Cappelli G
      • Santucci M
      • et al.
      Expression of CCR5 is increased in human monocyte-derived macrophages and alveolar macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis infection.
      • Kunkel EJ
      • Boisvert J
      • Murphy K
      • et al.
      Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes.
      • Ma B
      • Liu W
      • Homer RJ
      • et al.
      Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodeling.
      • van Deventer HW
      • O'Connor Jr, W
      • Brickey WJ
      • Aris RM
      • Ting JP
      • Serody JS
      C-C chemokine receptor 5 on stromal cells promotes pulmonary metastasis.
      Inhibition of RANTES signaling significantly abrogates tumor invasion, suggesting that RANTES is required for invasion in TGF-² type II receptor repressed lung AC cells (Fig. 1C). The clinical significance of this pathway is further supported by the finding that tumor expression of RANTES and CCR5 in lung AC is associated with patient survival.
      • Borczuk AC
      • Papanikolaou N
      • Toonkel RL
      • et al.
      Lung adenocarcinoma invasion in TGFbetaRII-deficient cells is mediated by CCL5/RANTES.
      Small molecule inhibitors of CCR5 may have the potential to treat and prevent lung AC. Taken together, these studies illustrate how information gained from global expression profiling of tumors can be used to identify key pathways and genes mediating tumor growth, invasion, and metastasis.

      REFERENCES

        • Brambilla E
        • Travis WD
        • Colby TV
        • Corrin B
        • Shimosato Y
        The new World Health Organization classification of lung tumours.
        Eur Respir J. 2001; 18: 1059-1068
        • Rena O
        • Papalia E
        • Ruffini E
        • et al.
        Stage I pure bronchioloalveolar carcinoma: recurrences, survival and comparison with adenocarcinoma of the lung.
        Eur J Cardiothorac Surg. 2003; 23: 409-414
        • Nakayama H
        • Yamada K
        • Saito H
        • et al.
        Sublobar resection for patients with peripheral small adenocarcinomas of the lung: surgical outcome is associated with features on computed tomographic imaging.
        Ann Thorac Surg. 2007; 84: 1675-1679
        • Henschke CI
        • McCauley DI
        • Yankelevitz DF
        • et al.
        Early Lung Cancer Action Project: overall design and findings from baseline screening.
        Lancet. 1999; 354: 99-105
        • Swensen SJ
        • Jett JR
        • Sloan JA
        • et al.
        Screening for lung cancer with low-dose spiral computed tomography.
        Am J Respir Crit Care Med. 2002; 165: 508-513
        • Maeshima AM
        • Niki T
        • Maeshima A
        • Yamada T
        • Kondo H
        • Matsuno Y
        Modified scar grade: a prognostic indicator in small peripheral lung adenocarcinoma.
        Cancer. 2002; 95: 2546-2554
        • Sakurai H
        • Maeshima A
        • Watanabe S
        • et al.
        Grade of stromal invasion in small adenocarcinoma of the lung: histopathological minimal invasion and prognosis.
        Am J Surg Pathol. 2004; 28: 198-206
        • Suzuki K
        • Yokose T
        • Yoshida J
        • et al.
        Prognostic significance of the size of central fibrosis in peripheral adenocarcinoma of the lung.
        Ann Thorac Surg. 2000; 69: 893-897
        • Yim J
        • Zhu LC
        • Chiriboga L
        • Watson HN
        • Goldberg JD
        • Moreira AL
        Histologic features are important prognostic indicators in early stages lung adenocarcinomas.
        Mod Pathol. 2007; 20: 233-241
        • Yokose T
        • Suzuki K
        • Nagai K
        • Nishiwaki Y
        • Sasaki S
        • Ochiai A
        Favorable and unfavorable morphological prognostic factors in peripheral adenocarcinoma of the lung 3 cm or less in diameter.
        Lung Cancer. 2000; 29: 179-188
        • Terasaki H
        • Niki T
        • Matsuno Y
        • et al.
        Lung adenocarcinoma with mixed bronchioloalveolar and invasive components: clinicopathological features, subclassification by extent of invasive foci, and immunohistochemical characterization.
        Am J Surg Pathol. 2003; 27: 937-951
        • Borczuk AC
        • Qian F
        • Kazeros A
        • et al.
        Invasive size is an independent predictor of survival in pulmonary adenocarcinoma.
        Am J Surg Pathol. 2009; 33: 462-469
        • Fidler IJ
        The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited.
        Nat Rev Cancer. 2003; 3: 453-458
        • Bissell MJ
        • Radisky D
        Putting tumours in context.
        Nat Rev Cancer. 2001; 1: 46-54
        • Elenbaas B
        • Weinberg RA
        Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation.
        Exp Cell Res. 2001; 264: 169-184
        • Borczuk AC
        • Kim HK
        • Yegen HA
        • Friedman RA
        • Powell CA
        Lung adenocarcinoma global profiling identifies type II transforming growth factor-beta receptor as a repressor of invasiveness.
        Am J Respir Crit Care Med. 2005; 172: 729-737
        • Takeuchi T
        • Tomida S
        • Yatabe Y
        • et al.
        Expression profile-defined classification of lung adenocarcinoma shows close relationship with underlying major genetic changes and clinicopathologic behaviors.
        J Clin Oncol. 2006; 24: 1679-1688
        • Motoi N
        • Szoke J
        • Riely GJ
        • et al.
        Lung adenocarcinoma: modification of the 2004 WHO mixed subtype to include the major histologic subtype suggests correlations between papillary and micropapillary adenocarcinoma subtypes, EGFR mutations and gene expression analysis.
        Am J Surg Pathol. 2008; 32: 810-827
        • Beer DG
        • Kardia SL
        • Huang CC
        • et al.
        Gene-expression profiles predict survival of patients with lung adenocarcinoma.
        Nat Med. 2002; 8: 816-824
        • Director's Challenge Consortium for the Molecular Classification of Lung Adenocarcinoma
        • Shedden K
        • Taylor JM
        • Enkemann SA
        • et al.
        Gene expression-based survival prediction in lung adenocarcinoma: a multi-site, blinded validation study.
        Nat Med. 2008; 14: 822-827
        • Ijichi H
        • Chytil A
        • Gorska AE
        • et al.
        Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression.
        Genes Dev. 2006; 20: 3147-3160
        • Lu SL
        • Herrington H
        • Reh D
        • et al.
        Loss of transforming growth factor-beta type II receptor promotes metastatic head-and-neck squamous cell carcinoma.
        Genes Dev. 2006; 20: 1331-1342
        • Munoz NM
        • Upton M
        • Rojas A
        • et al.
        Transforming growth factor beta receptor type II inactivation induces the malignant transformation of intestinal neoplasms initiated by Apc mutation.
        Cancer Res. 2006; 66: 9837-9844
        • Siegel PM
        • Massague J
        Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer.
        Nat Rev Cancer. 2003; 3: 807-821
        • Yoshinaga K
        • Obata H
        • Jurukovski V
        • et al.
        Perturbation of transforming growth factor (TGF)-beta1 association with latent TGF-beta binding protein yields inflammation and tumors.
        Proc Natl Acad Sci USA. 2008; 105: 18758-18763
        • Massague J
        How cells read TGF-beta signals.
        Nat Rev Mol Cell Biol. 2000; 1: 169-178
        • Derynck R
        • Akhurst RJ
        • Balmain A
        TGF-beta signaling in tumor suppression and cancer progression.
        Nat Genet. 2001; 29: 117-129
        • Elliott RL
        • Blobe GC
        Role of transforming growth factor beta in human cancer.
        J Clin Oncol. 2005; 23: 2078-2093
        • Tang B
        • Vu M
        • Booker T
        • et al.
        TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression.
        J Clin Invest. 2003; 112: 1116-1124
        • Roberts AB
        • Wakefield LM
        The two faces of transforming growth factor beta in carcinogenesis.
        Proc Natl Acad Sci USA. 2003; 100: 8621-8623
        • Markowitz S
        • Wang J
        • Myeroff L
        • et al.
        Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability.
        Science. 1995; 268: 1336-1338
        • Kang SH
        • Bang YJ
        • Im YH
        • et al.
        Transcriptional repression of the transforming growth factor-beta type I receptor gene by DNA methylation results in the development of TGF-beta resistance in human gastric cancer.
        Oncogene. 1999; 18: 7280-7286
        • Bierie B
        • Moses HL
        TGF-beta and cancer.
        Cytokine Growth Factor Rev. 2006; 17: 29-40
        • Levy L
        • Hill CS
        Alterations in components of the TGF-beta superfamily signaling pathways in human cancer.
        Cytokine Growth Factor Rev. 2006; 17: 41-58
        • Kim SJ
        • Im YH
        • Markowitz SD
        • Bang YJ
        Molecular mechanisms of inactivation of TGF-beta receptors during carcinogenesis.
        Cytokine Growth Factor Rev. 2000; 11: 159-168
        • Biswas S
        • Chytil A
        • Washington K
        • et al.
        Transforming growth factor beta receptor type II inactivation promotes the establishment and progression of colon cancer.
        Cancer Res. 2004; 64: 4687-4692
        • Grady WM
        • Rajput A
        • Myeroff L
        • et al.
        Mutation of the type II transforming growth factor-beta receptor is coincident with the transformation of human colon adenomas to malignant carcinomas.
        Cancer Res. 1998; 58: 3101-3104
        • Forrester E
        • Chytil A
        • Bierie B
        • et al.
        Effect of conditional knockout of the type II TGF-beta receptor gene in mammary epithelia on mammary gland development and polyomavirus middle T antigen induced tumor formation and metastasis.
        Cancer Res. 2005; 65: 2296-2302
        • Gobbi H
        • Dupont WD
        • Simpson JF
        • et al.
        Transforming growth factor-beta and breast cancer risk in women with mammary epithelial hyperplasia.
        J Natl Cancer Inst. 1999; 91: 2096-2101
        • Hougaard S
        • Norgaard P
        • Abrahamsen N
        • Moses HL
        • Spang-Thomsen M
        • Skovgaard Poulsen H
        Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines.
        Br J Cancer. 1999; 79: 1005-1011
        • Damstrup L
        • Rygaard K
        • Spang-Thomsen M
        • Skovgaard Poulsen H
        Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines.
        Br J Cancer. 1993; 67: 1015-1021
        • de Jonge RR
        • Garrigue-Antar L
        • Vellucci VF
        • Reiss M
        Frequent inactivation of the transforming growth factor beta type II receptor in small-cell lung carcinoma cells.
        Oncol Res. 1997; 9: 89-98
        • Kim WS
        • Park C
        • Hong SK
        • Park BK
        • Kim HS
        • Park K
        Microsatellite instability (MSI) in non-small cell lung cancer (NSCLC) is highly associated with transforming growth factor-beta type II receptor (TGF-beta RII) frameshift mutation.
        Anticancer Res. 2000; 20: 1499-1502
        • Kim TK
        • Mo EK
        • Yoo CG
        • et al.
        Alteration of cell growth and morphology by overexpression of transforming growth factor beta type II receptor in human lung adenocarcinoma cells.
        Lung Cancer. 2001; 31: 181-191
        • Park C
        • Kim WS
        • Choi Y
        • Kim H
        • Park K
        Effects of transforming growth factor beta (TGF-beta) receptor on lung carcinogenesis.
        Lung Cancer. 2002; 38: 143-147
        • Anumanthan G
        • Halder SK
        • Osada H
        • et al.
        Restoration of TGF-beta signalling reduces tumorigenicity in human lung cancer cells.
        Br J Cancer. 2005; 93: 1157-1167
        • Kang Y
        • Prentice MA
        • Mariano JM
        • et al.
        Transforming growth factor-beta 1 and its receptors in human lung cancer and mouse lung carcinogenesis.
        Exp Lung Res. 2000; 26: 685-707
        • Zhang HT
        • Chen XF
        • Wang MH
        • et al.
        Defective expression of transforming growth factor beta receptor type II is associated with CpG methylated promoter in primary non-small cell lung cancer.
        Clin Cancer Res. 2004; 10: 2359-2367
        • Zhao J
        • Liu Z
        • Li W
        • Liu X
        • Chen XF
        • Zhang HT
        Infrequently methylated event at sites −362 to −142 in the promoter of TGF beta R1 gene in non-small cell lung cancer.
        J Cancer Res Clin Oncol. 2008; 134: 919-925
        • Zhang HT
        • Fei QY
        • Chen F
        • et al.
        Mutational analysis of the transforming growth factor beta receptor type I gene in primary non-small cell lung cancer.
        Lung Cancer. 2003; 40: 281-287
        • Kang HG
        • Chae MH
        • Park JM
        • et al.
        Polymorphisms in TGF-beta1 gene and the risk of lung cancer.
        Lung Cancer. 2006; 52: 1-7
        • Park KH
        • Lo Han SG
        • Whang YM
        • et al.
        Single nucleotide polymorphisms of the TGFB1 gene and lung cancer risk in a Korean population.
        Cancer Genet Cytogenet. 2006; 169: 39-44
        • Barthelemy-Brichant N
        • David JL
        • Bosquee L
        • et al.
        Increased TGFbeta1 plasma level in patients with lung cancer: potential mechanisms.
        Eur J Clin Invest. 2002; 32: 193-198
        • Lee JC
        • Lee KM
        • Kim DW
        • Heo DS
        Elevated TGF-beta1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients.
        J Immunol. 2004; 172: 7335-7340
        • Domagala-Kulawik J
        • Hoser G
        • Safianowska A
        • Grubek-Jaworska H
        • Chazan R
        Elevated TGF-beta1 concentration in bronchoalveolar lavage fluid from patients with primary lung cancer.
        Arch Immunol Ther Exp (Warsz). 2006; 54: 143-147
        • Bruna A
        • Darken RS
        • Rojo F
        • et al.
        High TGFbeta-Smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene.
        Cancer Cell. 2007; 11: 147-160
        • Hasegawa Y
        • Takanashi S
        • Kanehira Y
        • Tsushima T
        • Imai T
        • Okumura K
        Transforming growth factor-beta1 level correlates with angiogenesis, tumor progression, and prognosis in patients with nonsmall cell lung carcinoma.
        Cancer. 2001; 91: 964-971
        • Saito H
        • Tsujitani S
        • Oka S
        • et al.
        The expression of transforming growth factor-beta1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma.
        Cancer. 1999; 86: 1455-1462
        • Tsushima H
        • Kawata S
        • Tamura S
        • et al.
        High levels of transforming growth factor beta 1 in patients with colorectal cancer: association with disease progression.
        Gastroenterology. 1996; 110: 375-382
        • Wikstrom P
        • Stattin P
        • Franck-Lissbrant I
        • Damber JE
        • Bergh A
        Transforming growth factor beta1 is associated with angiogenesis, metastasis, and poor clinical outcome in prostate cancer.
        Prostate. 1998; 37: 19-29
        • Oft M
        • Heider KH
        • Beug H
        TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis.
        Curr Biol. 1998; 8: 1243-1252
        • Deckers M
        • van Dinther M
        • Buijs J
        • et al.
        The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells.
        Cancer Res. 2006; 66: 2202-2209
        • Muraoka-Cook RS
        • Kurokawa H
        • Koh Y
        • et al.
        Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors.
        Cancer Res. 2004; 64: 9002-9011
        • Muraoka-Cook RS
        • Shin I
        • Yi JY
        • et al.
        Activated type I TGFbeta receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression.
        Oncogene. 2006; 25: 3408-3423
        • Siegel PM
        • Shu W
        • Cardiff RD
        • Muller WJ
        • Massague J
        Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis.
        Proc Natl Acad Sci USA. 2003; 100: 8430-8435
        • Tian F
        • Byfield SD
        • Parks WT
        • et al.
        Smad-binding defective mutant of transforming growth factor beta type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.
        Cancer Res. 2004; 64: 4523-4530
        • Tian F
        • DaCosta Byfield S
        • Parks WT
        • et al.
        Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.
        Cancer Res. 2003; 63: 8284-8292
        • Bandyopadhyay A
        • Agyin JK
        • Wang L
        • et al.
        Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor.
        Cancer Res. 2006; 66: 6714-6721
        • Yang YA
        • Dukhanina O
        • Tang B
        • et al.
        Lifetime exposure to a soluble TGF-beta antagonist protects mice against metastasis without adverse side effects.
        J Clin Invest. 2002; 109: 1607-1615
        • Biswas S
        • Guix M
        • Rinehart C
        • et al.
        Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression.
        J Clin Invest. 2007; 117: 1305-1313
        • Nam JS
        • Terabe M
        • Mamura M
        • et al.
        An anti-transforming growth factor beta antibody suppresses metastasis via cooperative effects on multiple cell compartments.
        Cancer Res. 2008; 68: 3835-3843
        • Saji H
        • Nakamura H
        • Awut I
        • et al.
        Significance of expression of TGF-beta in pulmonary metastasis in non-small cell lung cancer tissues.
        Ann Thorac Cardiovasc Surg. 2003; 9: 295-300
        • Rojas A
        • Padidam M
        • Cress D
        • Grady WM
        TGF-beta receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-beta.
        Biochim Biophys Acta. 2009; 1793: 1165-1173
        • Yang L
        • Huang J
        • Ren X
        • et al.
        Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis.
        Cancer Cell. 2008; 13: 23-35
        • Bhowmick NA
        • Zent R
        • Ghiassi M
        • McDonnell M
        • Moses HL
        Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38mapk and epithelial plasticity.
        J Biol Chem. 2001; 276: 46707-46713
        • Fraziano M
        • Cappelli G
        • Santucci M
        • et al.
        Expression of CCR5 is increased in human monocyte-derived macrophages and alveolar macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis infection.
        AIDS Res Hum Retroviruses. 1999; 15: 869-874
        • Kunkel EJ
        • Boisvert J
        • Murphy K
        • et al.
        Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes.
        Am J Pathol. 2002; 160: 347-355
        • Ma B
        • Liu W
        • Homer RJ
        • et al.
        Role of CCR5 in the pathogenesis of IL-13-induced inflammation and remodeling.
        J Immunol. 2006; 176: 4968-4978
        • van Deventer HW
        • O'Connor Jr, W
        • Brickey WJ
        • Aris RM
        • Ting JP
        • Serody JS
        C-C chemokine receptor 5 on stromal cells promotes pulmonary metastasis.
        Cancer Res. 2005; 65: 3374-3379
        • Borczuk AC
        • Papanikolaou N
        • Toonkel RL
        • et al.
        Lung adenocarcinoma invasion in TGFbetaRII-deficient cells is mediated by CCL5/RANTES.
        Oncogene. 2008; 27: 557-564
        • Borczuk AC
        • Toonkel RL
        • Powell CA
        Genomics of lung cancer.
        Proc Am Thorac Soc. 2009; 6: 152-158