Metastasis occurs when genetically unstable cancer cells adapt to a tissue microenvironment that is distant from the primary tumor. This process involves both the selection of traits that are advantageous to cancer cells and the concomitant recruitment of traits in the tumor stroma that accommodate invasion by metastatic cells. Recent conceptual and technological advances promote our understanding of the origins and nature of cancer metastasis.

By means of in vivo selection, transcriptomic analysis, functional verification and clinical validation, here we identify a set of genes that marks and mediates breast cancer metastasis to the lungs. Some of these genes serve dual functions, providing growth advantages both in the primary tumour and in the lung microenvironment. Others contribute to aggressive growth selectively in the lung. Many encode extracellular proteins and are of previously unknown relevance to cancer metastasis.Metastasis is frequently a final and fatal step in the progression of solid malignancies. Tumour cell intravasation, survival in circulation, extravasation into a distant organ, angiogenesis and uninhibited growth constitute the metastatic process 1 . The molecular requirements for some of these steps may be tissue specific. Indeed, the proclivity that tumours have for specific organs, such as breast carcinomas for bone and lung, was noted more than a century ago 2 .The identity and time of onset of the changes that endow tumour cells with these metastatic functions are largely unknown and are a subject of debate. It is believed that genomic instability generates large-scale cellular heterogeneity within tumour populations, from which rare cellular variants with augmented metastatic abilities evolve through a darwinian selection process 2,3 . Work on experimental metastasis with tumour cell lines has demonstrated that reinjection of metastatic cell populations can lead to enrichment in the metastatic phenotype 4-6 . Recently, however, the existence of genes expressed by rare cellular variants that specifically mediate metastasis has been challenged 7 . Transcriptomic profiling of primary human carcinomas has identified gene expression patterns that, when present in the bulk primary tumour population, predict a poor prognosis for patients 8-10 . The existence of such signatures has been interpreted to mean that genetic lesions acquired early in tumor-igenesis are sufficient for the metastatic process, and that consequently no metastasis-specific genes exist. However, it is unclear whether these genes predicting metastatic recurrence are also functional mediators.The lungs and bones are frequent sites of breast cancer metastasis, and metastases to these sites differ in terms of their evolution, treatment, morbidity and mortality 11 . Reasoning that each organ places different demands on circulating cancer cells for the establishment of metastases, Selection of cells metastatic to the lungsThe cell line MDA-MB-231 was derived from the pleural effusion of a breast cancer patient suffering from widespread metastasis years after removal of her primary tumour 12 . Individual MDA-MB-231 cells grown and tested as single-cell-derived progenies (SCPs) have distinct metastatic abilities and tissue tropisms 13 despite having similar expression levels of genes constituting a validated Rosetta-type poor prognosis signature 9 ( Supplementary Fig. S1). These different meta-static behaviours, including different tropisms to bone and lung, ...

Metastasis entails numerous biological functions that collectively enable cancerous cells from a primary site to disseminate and overtake distant organs. Using genetic and pharmacological approaches, we show that the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2, when expressed in human breast cancer cells, collectively facilitate the assembly of new tumour blood vessels, the release of tumour cells into the circulation, and the breaching of lung capillaries by circulating tumour cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.

TGF-␤ can signal by means of Smad transcription factors, which are quintessential tumor suppressors that inhibit cell proliferation, and by means of Smad-independent mechanisms, which have been implicated in tumor progression. Although Smad mutations disable this tumor-suppressive pathway in certain cancers, breast cancer cells frequently evade the cytostatic action of TGF-␤ while retaining Smad function. Through immunohistochemical analysis of human breast cancer bone metastases and functional imaging of the Smad pathway in a mouse xenograft model, we provide evidence for active Smad signaling in human and mouse bonemetastatic lesions. Genetic depletion experiments further demonstrate that Smad4 contributes to the formation of osteolytic bone metastases and is essential for the induction of IL-11, a gene implicated in bone metastasis in this mouse model system. Activator protein-1 is a key participant in Smad-dependent transcriptional activation of IL-11 and its overexpression in bone-metastatic cells. Our findings provide functional evidence for a switch of the Smad pathway, from tumor-suppressor to prometastatic, in the development of breast cancer bone metastasis.IL-11 ͉ Smad4 ͉ TGF-␤ T GF-␤ plays a crucial role as a growth-inhibitory cytokine in many tissues (1, 2). The cytostatic effect of TGF-␤ is mediated by a serine͞threonine kinase receptor complex that phosphorylates Smad2 and Smad3, which then translocate into the nucleus and bind Smad4 to generate transcriptional regulatory complexes (3). SMAD4 (also known as Deleted in Pancreatic Carcinoma locus 4 or DPC4) and, to a lesser extent, SMAD2 suffer mutational inactivation in a proportion of pancreatic and colon cancers (1, 2). However, tumor cells that evade this antiproliferative control by other mechanisms may display an altered sensitivity to TGF-␤ and undergo tumorigenic progression in response to this cytokine (1, 2). Patients whose pancreatic or colon tumors express TGF-␤ receptors fare less well than those with low or absent TGF-␤ receptor expression in the tumor (4). In mouse models of breast cancer, TGF-␤ signaling promotes lung (5, 6) and bone metastasis (7). In the case of osteolytic bone metastasis by breast cancer cells, it has been proposed that TGF-␤ released from the decaying bone matrix stimulates neighboring tumor cells, establishing a vicious cycle that exacerbates the growth of the metastatic lesion (8).The TGF-␤ signaling mechanisms that foster metastasis in human cancer are an important open question and a subject of debate. Because Smad factors are quintessential tumor suppressors, the basis for the protumorigenic effects of TGF-␤ has been sought in the Smad-independent signaling pathways that may be triggered by TGF-␤. Results obtained by means of overexpression of dominant negative mutant components of the Rho pathway (9, 10) or pharmacologic inhibitors of p38 mitogen-activated protein kinase (11, 12) have implicated these pathways in the proinvasive and metastatic effects of TGF-␤ in transformed cells. In contrast, results obta...

We used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in immunodeficient mice. Individual cells from a population established in culture from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis. Single-cell progenies derived from this population exhibited markedly different abilities to metastasize to the bone, lung, or adrenal medulla, which suggests that metastases to different organs have different requirements. Transcriptomic profiling revealed that these different single-cell progenies similarly express a previously described "poor-prognosis" gene expression signature. Unsupervised classification using the transcriptomic data set supported the hypothesis that organ-specific metastasis by breast cancer cells is controlled by metastasis-specific genes that are separate from a general poor-prognosis gene expression signature. Furthermore, by using a gene expression signature associated with the ability of these cells to metastasize to bone, we were able to distinguish primary breast carcinomas that preferentially metastasized to bone from those that preferentially metastasized elsewhere. These results suggest that the bone-specific metastatic phenotypes and gene expression signature identified in a mouse model may be clinically relevant.

Summary DNA Polymerase theta (Pol θ) mediated end-joining (TMEJ) has been implicated in repair of chromosome breaks, but its cellular mechanism and role relative to canonical repair pathways is poorly understood. We show it accounts for most repair associated with microhomologies, and is made efficient by coupling a microhomology search to removal of nonhomologous tails and microhomology-primed synthesis across broken ends. In contrast to nonhomologous end-joining (NHEJ), TMEJ efficiently repairs end structures expected after aborted homology-directed repair (5′ to 3′ resected ends) or replication fork collapse. It typically does not compete with canonical repair pathways, but in NHEJ-deficient cells is engaged more frequently and protects against translocation. Cell viability is also severely impaired upon combined deficiency in Pol θ and a factor that antagonizes end resection (Ku or 53BP1). TMEJ thus helps sustain cell viability and genome stability by rescuing chromosome break repair when resection is misregulated or NHEJ is compromised.

The association between large tumor size and metastatic risk in a majority of clinical cancers has led to questions as to whether these observations are causally related or whether one is simply a marker for the other. This is partly due to an uncertainty about how metastasis-promoting gene expression changes can arise in primary tumors. We investigated this question through the analysis of a previously defined ''lung metastasis gene-expression signature'' (LMS) that mediates experimental breast cancer metastasis selectively to the lung and is expressed by primary human breast cancer with a high risk for developing lung metastasis. Experimentally, we demonstrate that the LMS promotes primary tumor growth that enriches for LMS ؉ cells, and it allows for intravasation after reaching a critical tumor size. Clinically, this corresponds to LMS ؉ tumors being larger at diagnosis compared with LMS ؊ tumors and to a marked rise in the incidence of metastasis after LMS ؉ tumors reach 2 cm. Patients with LMS-expressing primary tumors selectively fail in the lung compared with the bone or other visceral sites and have a worse overall survival. The mechanistic linkage between metastasis gene expression, accelerated tumor growth, and likelihood of metastatic recurrence provided by the LMS may help to explain observations of prognostic gene signatures in primary cancer and how tumor growth can both lead to metastasis and be a marker for cells destined to metastasize.cancer ͉ genomics ͉ oncogenesis T he consistent association of large tumor size, rapid growth rate, and metastatic risk in a majority of cases of clinical cancer suggests that the molecular bases of these phenomena may be linked (1-3). However, the nature of this link remains unresolved. Conventional models of metastasis envision rare metastatically competent variants emerging by chance as primary tumors grow, causally linking growth with likelihood of metastatic relapse (4, 5). In this view, genes that control primary tumor growth operate independently of stochastically acquired metastasis genes. Alternative models posit that prometastatic gene expression events are acquired early during tumorigenesis and may overlap with the genes that promote primary tumor growth, making tumor size a marker for metastatic risk (6). These alternative models form a teleological basis for using gene expression signatures from primary tumors to forecast whether patients are at high risk for micrometastatic disease. However, despite several reports on the success of gene signatures from primary tumors to predict development of distant spread (7-12), tumor size remains an independent prognostic factor on multivariate analysis (9). Thus, to what degree conventional versus alternative models can explain the acquisition of a metastatic phenotype remains unclear.One of the difficulties in addressing the fundamental question on how metastasis gene expression events are acquired relates to the genetically complex nature of the phenotype itself. It has long been believed that there are nume...

Purpose: To identify a profile of circulating tumor human papilloma virus (HPV) DNA (ctHPVDNA) clearance kinetics that is associated with disease control after chemoradiotherapy (CRT) for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Experimental Design: A multi-institutional prospective biomarker trial was conducted in 103 patients with (i) p16positive OPSCC, (ii) M0 disease, and (iii) receipt of definitive CRT. Blood specimens were collected at baseline, weekly during CRT, and at follow-up visits. Optimized multianalyte digital PCR assays were used to quantify ctHPVDNA (types 16/18/31/33/35) in plasma. A control cohort of 55 healthy volunteers and 60 patients with non-HPV-associated malignancy was also analyzed. Results: Baseline plasma ctHPVDNA had high specificity (97%) and high sensitivity (89%) for detecting newly diagnosed HPV-associated OPSCC. Pretreatment ctHPV16DNA copy number correlated with disease burden, tumor HPV copy number, and HPV integration status. We define a ctHPV16DNA favorable clearance profile as having high baseline copy number (>200 copies/mL) and >95% clearance of ctHPV16DNA by day 28 of CRT. Nineteen of 67 evaluable patients had a ctHPV16DNA favorable clearance profile, and none had persistent or recurrent regional disease after CRT. In contrast, patients with adverse clinical risk factors (T4 or >10 pack years) and an unfavorable ctHPV16DNA clearance profile had a 35% actuarial rate of persistent or recurrent regional disease after CRT (P ¼ 0.0049). Conclusions: A rapid clearance profile of ctHPVDNA may predict likelihood of disease control in patients with HPVassociated OPSCC patients treated with definitive CRT and may be useful in selecting patients for deintensified therapy.