SUMMARY Cancer therapy exerts a strong selection pressure that shapes tumor evolution, yet our knowledge of how tumors change during treatment is limited. Here we report the analysis of cellular heterogeneity for genetic and phenotypic features and their spatial distribution in breast tumors pre- and post-neoadjuvant chemotherapy. We found that intratumor genetic diversity was tumor subtype-specific and it did not change during treatment in tumors with partial or no response. However, lower pre-treatment genetic diversity was significantly associated with complete pathologic response. In contrast, phenotypic diversity was different between pre- and post-treatment samples. We also observed significant changes in the spatial distribution of cells with distinct genetic and phenotypic features. We used these experimental data to develop a stochastic computational model to infer tumor growth patterns and evolutionary dynamics. Our results highlight the importance of integrated analysis of genotypes and phenotypes of single cells in intact tissues to predict tumor evolution.

Nerve fibers accompany blood and lymphatic vessels all over the body. An extensive amount of knowledge has been obtained with regard to tumor angiogenesis and tumor lymphangiogenesis, yet little is known about the potential biological effects of "neoneurogenesis". Cancer cells can exploit the advantage of the factors released by the nerve fibers to generate a positive microenvironment for cell survival and proliferation. At the same time, they can stimulate the formation of neurites by secreting neurotrophic factors and axon guidance molecules. The neuronal influence on the biology of a neoplasm was initially described several decades ago. Since then, an increasing amount of experimental evidence strongly suggests the existence of reciprocal interactions between cancer cells and nerves in humans. Moreover, researchers have been able to demonstrate a crosstalk between cancer cells and nerve fibers as a strategy for survival. Despite all these evidence, a lot remains to be done in order to clarify the role of neurotransmitters, neuropeptides, and their associated receptor-initiated signaling pathways in the development and progression of cancer, and response to therapy. A global-wide characterization of the neurotransmitters or neuropeptides present in the tumor microenvironment would provide insights into the real biological influences of the neuronal tissue on tumor progression. This review is intended to discuss our current understanding of neurosignaling in cancer and its potential implications on cancer prevention and therapy. The review will focus on the soluble factors released by cancer cells and nerve endings, their biological effects and their potential relevance in the treatment of cancer.

In cancer cachexia both cardiac and skeletal muscle suffer an important protein mobilization as a result of increased proteolysis. Administration of the beta2-agonist formoterol to both rats and mice bearing highly cachectic tumors resulted in an important reversal of the muscle-wasting process. The anti-wasting effects of the drug were based on both an activation of the rate of protein synthesis and an inhibition of the rate of muscle proteolysis. Northern blot analysis revealed that formoterol treatment resulted in a decrease in the mRNA content of ubiquitin and proteasome subunits in gastrocnemius muscles; this, together with the decreased proteasome activity observed, suggest that the main anti-proteolytic action of the drug may be based on an inhibition of the ATP-ubiquitin-dependent proteolytic system. Interestingly, the beta2-agonist was also able to diminish the increased rate of muscle apoptosis (measured as DNA laddering as well as caspase-3 activity) present in tumor-bearing animals. The present results indicate that formoterol exerted a selective, powerful protective action on heart and skeletal muscle by antagonizing the enhanced protein degradation that characterizes cancer cachexia, and it could be revealed as a potential therapeutic tool in pathologic states wherein muscle protein hypercatabolism is a critical feature such as cancer cachexia or other wasting diseases.

BackgroundThe efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells.Principal FindingsFor this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53+/+, RHCT116 p53−/−) from the parental HT29, HCT116 p53+/+ and HCT116 p53−/− colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53−/− but not in the RHCT116 p53+/+. Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53−/− but not in the RHCT116 p53+/+. Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells.ConclusionsTaking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals.

ERBB receptor transmodulation by heterologous G-protein-coupled receptors (GPCR) generates functional diversity in signal transduction. Tachykinins are neuropeptides and proinflammatory cytokines that promote cell survival and cancer progression by activating several GPCRs. In this work, we found that the pain-associated tachykinin Substance P (SP) contributes to persistent transmodulation of the ERBB receptors, EGFR and HER2, in breast cancer, acting to enhance malignancy and therapeutic resistance. SP and its high-affinity receptor NK-1R were highly expressed in HER2 þ primary breast tumors (relative to the luminal and triple-negative subtypes) and were overall correlated with poor prognosis factors. In breast cancer cell lines and primary cultures derived from breast cancer samples, we found that SP could activate HER2. Conversely, RNA interference-mediated attenuation of NK-1R, or its chemical inhibition, or suppression of overall GPCR-mediated signaling, all strongly decreased steady-state expression of EGFR and HER2, establishing that their basal activity relied upon transdirectional activation by GPCR. Thus, SP exposure affected cellular responses to anti-ERBB therapies. Our work reveals an important oncogenic cooperation between NK-1R and HER2, thereby adding a novel link between inflammation and cancer progression that may be targetable by SP antagonists that have been clinically explored. Cancer Res; 73(21); 6424-34. Ó2013 AACR.

NK1 is a tachykinin receptor highly relevant to tumorigenesis and metastasis development in breast cancer and other carcinomas. Despite the substantial efforts done to develop potent NK1 receptor antagonists, none of these antagonists had shown good antitumor activity in clinical trials. Now, we have tested the effect of inhibition of the neuropeptide Substance P (SP), a NK1 ligand, as a potential therapeutic approach in cancer. We found that the inhibition of SP with antibodies strongly inhibit cell growth and induce apoptosis in breast, colon, and prostate cancer cell lines. These effects were accompained by a decrease in the mitogen-activated kinase singaling pathway. Interestingly, in some cell lines SP abrogation decreased the steady state of Her2 and EGFR, suggesting that SP-mediated signaling is important for the basal activity of these ErbB receptors. In consequence, we observed a blockade of the cell cycle progression and the inhibition of several cell cycle-related proteins including mTOR. SP inhibition also induced cell death in cell lines resistant to Lapatinib and Trastuzumab that have increased levels of active Her2, suggesting that this therapeutic approach could be also effective for those cancers resistant to current anti-ErbB therapies. Thus, we propose a new therapeutic strategy for those cancers that express NK1 receptor and/or other tachykinin receptors, based in the immuno-blockade of the neuropeptide SP.

Purpose: Despite the therapeutic success of existing HER2-targeted therapies, tumors invariably relapse. This study aimed at identifying new mechanisms responsible for HER2-targeted therapy resistance. Experimental Design: We have used a platform of HER2-targeted therapy–resistant cell lines and primary cultures of healthy and tumor-associated fibroblasts (TAF) to identify new potential targets related to tumor escape from anti-HER2 therapies. Results: We have shown that TAFs promote resistance to HER2-targeted therapies. TAFs produce and secrete high levels of FGF5, which induces FGFR2 activation in the surrounding breast cancer cells. FGFR2 transactivates HER2 via c-Src, leading to resistance to HER2-targeted therapies. In vivo, coinoculating nonresistant cell lines with TAFs results in more aggressive and resistant tumors. Resistant cells activate fibroblasts and secrete FGFR ligands, creating a positive feedback loop that fuels resistance. FGFR2 inhibition not only inhibits HER2 activation, but also induces apoptosis in cells resistant to HER2-targeted therapies. In vivo, inhibitors of FGFR2 reverse resistance and resensitize resistant cells to HER2-targeted therapies. In HER2 patients' samples, α-SMA, FGF5, and FGFR2 contribute to poor outcome and correlate with c-Src activation. Importantly, expression of FGF5 and phospho-HER2 correlated with a reduced pathologic complete response rate in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab, which highlights the significant role of TAFs/FGF5 in HER2 breast cancer progression and resistance. Conclusions: We have identified the TAF/FGF5/FGFR2/c-Src/HER2 axis as an escape pathway responsible for HER2-targeted therapy resistance in breast cancer, which can be reversed by FGFR inhibitors.

Implantation of the Yoshida AH-130 ascites hepatoma to rats resulted in a decrease in muscle weight 7 days after the inoculation of the tumor. These changes were associated with increases in the mRNA content for both peroxisome proliferator-activated receptor (PPAR) gamma and PPAR delta in skeletal muscle. The increase in gene expression for these transcription factors was related to increases in the expression of several genes involved in fatty acid transport, activation, and oxidation. Tumor burden also resulted in increases in PPAR gamma coactivator-1 alpha gene expression and pyruvate dehydrogenase kinase 4. All these changes in lipid metabolism genes suggest that a metabolic shift occurs in skeletal muscle of tumor-bearing rats toward a more oxidative phenotype. Formoterol treatment to tumor-bearing rats resulted in an amelioration of all the changes observed as a result of tumor burden. Administration of this beta(2)-adrenergic agonist also resulted in a decrease in mRNA content of muscle PPAR alpha, PPAR delta, and PPAR gamma, as well as in mRNA levels of many of the genes involved in both lipid and mitochondrial metabolism. All these results suggest an involvement of the different PPARs as transcription factors related with muscle wasting and also indicate that a possible mode of action of the anticachectic compound formoterol may involve a normalization of the levels of these transcription factors.