Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3-mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA-inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple-negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple-negative breast cancers. In MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells, ALDH1A3 and RA increased expression of RA-inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA-MB-231 and MDA-MB-435 cells, but decreasing tumor growth of MDA-MB-468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA-MB-231 and MDA-MB-468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5-aza-2'deoxycytidine restored uniform RA-inducibility of RARE-containing HOXA1 and MUC4 in MDA-MB-231 and MDA-MB-468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA-induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.

INTRODUCTION: Incisional biopsies, including the diagnostic core needle biopsy (CNB), routinely performed before surgical excision of breast cancer tumors are hypothesized to increase the risk of metastatic disease. In this study, we experimentally determined whether CNB of breast cancer tumors results in increased distant metastases and examine important resultant changes in the primary tumor and tumor microenvironment associated with this outcome. METHOD: To evaluate the effect of CNB on metastasis development, we implanted murine mammary 4T1 tumor cells in BALB/c mice and performed CNB on palpable tumors in half the mice. Subsequently, emulating the human scenario, all mice underwent complete tumor excision and were allowed to recover, with attendant metastasis development. Tumor growth, lung metastasis, circulating tumor cell (CTC) levels, variation in gene expression, composition of the tumor microenvironment, and changes in immunologic markers were compared in biopsied and non-biopsied mice. RESULTS: Mice with biopsied tumors developed significantly more lung metastases compared to non-biopsied mice. Tumors from biopsied mice contained a higher frequency of myeloid-derived suppressor cells (MDSCs) accompanied by reduced CD4 + T cells, CD8 + T cells, and macrophages, suggesting biopsy-mediated development of an increasingly immunosuppressive tumor microenvironment. We also observed a CNB-dependent up-regulation in the expression of SOX4, Ezh2, and other key epithelial-mesenchymal transition (EMT) genes, as well as increased CTC levels among the biopsy group. CONCLUSION: CNB creates an immunosuppressive tumor microenvironment, increases EMT, and facilitates release of CTCs, all of which likely contribute to the observed increase in development of distant metastases.

e21090 Background: Given the reported association between incisional procedures performed on cancer patients and subsequent increases in metastasis, as well as the established inverse relationship between metastasis and patient survival, this study establishes the extent to which incisional core needle biopsies (CNB) affects tumor growth and metastatic dissemination in two distinct breast cancer animal models. Methods: Using the chick embryo system (CES) and murine breast cancer model (MuBC)the impact of CNB on cancer metastases was evaluated. Human MDA-MB-231 and MDA-MB-435 cancer cells were used in the xenograft / CES, and murine 4T1 Breast cancer cells for the syngeneic MuBC . In each model, tumors were biopsied in half of the animals while the other half were left undisturbed (CES:n=40, MuBC:n=40). The impact of CNB on tumor growth, necrosis and metastases was assessed. Metastases levels in the CES was determined by quantitative PCR for human alu sequence DNA in chick tissue extracts. Metastatic burden in the MuBC model was evaluated by microscopic quantification of metastatic areas in sectioned and H-E stained mouse organs. Results: When biopsied and un-biopsied groups were compared, both models showed significant difference in pulmonary metastasis. MDA-MB-435 CES (p=0.025) and 4T1 MuBC (p=0.026). MDA-MB-231 CES showed no significant change in lung metastases in the CES but did however show increased Chorioallantoic Membrane (CAM) metastasis (p=0.018) and both cell lines showed statistically significant alteration in Liver metastasis, (MDA-MB-231 CES (p=0.006); MDA-MB-435 CES (p=0.004)). Interestingly only MDA-MB-435 (CES) tumor growth was significantly increased after CNB (p=0.002). Conclusions: These results offer the first experimental evidence that core needle biopsies result in an increased risk of metastatic dissemination and affect metastatic tropic behavior. They also reinforce the concept of a multi-step metastatic pathway and suggest involvement of extrinsic factors that influence the extravastion and intravasation steps. The clinical implications are considerable and follow-up studies examining potential mechanisms and countermeasures are urgently required

As a consequence of advances in digital diagnostic imaging, breast cancers are being diagnosed earlier in their development. These smaller tumors are less likely to be metastatic at time of discovery. The majority of newly diagnosed breast cancers in North America are confirmed using an incisional core needle biopsy (CNB). However an increasing number of studies highlight the pro-metastatic inflammatory and microenvironment changes occurring after incisional procedures on breast cancer tumors. This may account for reported higher incidences of lymph node metastases with increased risk of local recurrence and distant metastasis among patients undergoing incisional procedures. Evidence from our studies using a metastatic mouse model shows that incisional core needle biopsies lead to increased pulmonary metastasis. To study the mechanism(s) involved we monitored changes in circulating tumor cells (CTC), cytokine levels, variations in gene expression, microenvironment alteration and pulmonary metastatic burden, following CNB. Gene expression analysis by real-time qPCR showed increased TGF-β expression accompanied by SOX4 upregulation at 3 hours post biopsy (SOX4 is a Master regulator of EMT). This was followed, at 24 hours post biopsy, by significantly increased levels of the SOX4-associated Ezh2 gene and other EMT genes (ZEB2, SNAI2, SNAI3, and CDH2). Monitoring of CTCs over the same time frame showed them to be significantly increased in biopsied mice from 6 to 24 hours post biopsy, indicating continual CTC egress from biopsied tumors. Flow cytometry analysis of tumor microenvironment showed an immunosuppressive microenvironment with increased MDSC levels, reduced CD4+ and CD8+ T cells, macrophage and NKT cell. Interestingly, similar analysis of the Lung microenvironment 4 days later (day 7 post biopsy) revealed an inversion of the tumor situation, in the form of a significant uptick in Th1 type changes described by increased CD4+ and CD8+ type T cells, macrophages and NK cells, as well as increased TNF-β gene expression indicators corresponding to an early response to growth of metastases. We also compared H&E stained histological sections of lungs from biopsied and un-biopsied mice and recorded significant increase in pulmonary metastasis for the biopsied group. The findings of our study indicate that performance of CNB results in increased metastasis with immunomodulation of the tumor microenvironment and unregulated SOX4/TGF- β. Additional studies are needed to further elucidate the mechanism, and explore mitigating strategies to counter CNB related metastasis. Citation Format: Edward G. Mathenge, Cheryl Dean, Derek Clements, Ahmad Vagharkashani, Steffany Photopoulos, Krysta Coyle, Benjamin A. Malueth, Mike Giacomantonio, Anna Nunokawa, Julie Jordan, Shashi Gujar, Paola Marcato, Partick Lee, Carman A. Giacomantonio. Biopsy induced metastasis: Role of SOX4/TGF-β driven EMT and immunosuppressive microenvironment. [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 1665. doi:10.1158/1538-7445.AM2014-1665