Cancer metastasis is no longer viewed as a linear cascade of events but rather as a series of concurrent, partially overlapping processes, as successfully metastasizing cells assume new phenotypes while jettisoning older behaviors. The lack of a systemic understanding of this complex phenomenon has limited progress in developing treatments for metastatic disease. Because metastasis has traditionally been investigated in distinct physiological compartments, the integration of these complex and interlinked aspects remains a challenge for both systems-level experimental and computational modeling of metastasis. Here, we present some of the current perspectives on the complexity of cancer metastasis, the multiscale nature of its progression, and a systems-level view of the processes underlying the invasive spread of cancer cells. We also highlight the gaps in our current understanding of cancer metastasis as well as insights emerging from interdisciplinary systems biology approaches to understand this complex phenomenon.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that mediates adaptive responses to hypoxia. We demonstrate that lysosomal degradation of the HIF-1α subunit by chaperone-mediated autophagy (CMA) is a major regulator of HIF-1 activity. Pharmacological inhibitors of lysosomal degradation, such as bafilomycin and chloroquine, increased HIF-1α levels and HIF-1 activity, whereas activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased HIF-1α levels and HIF-1 activity. In contrast, macroautophagy inhibitors did not increase HIF-1 activity. Transcription factor EB, a master regulator of lysosomal biogenesis, also negatively regulated HIF-1 activity. HIF-1α interacts with HSC70 and LAMP2A, which are core components of the CMA machinery. Overexpression of HSC70 or LAMP2A decreased HIF-1α protein levels, whereas knockdown had the opposite effect. Finally, hypoxia increased the transcription of genes involved in CMA and lysosomal biogenesis in cancer cells. Thus, pharmacological and genetic approaches identify CMA as a major regulator of HIF-1 activity and identify interplay between autophagy and the response to hypoxia.

Metastasis involves critical interactions between cancer and stromal cells. Intratumoral hypoxia promotes metastasis through activation of hypoxia-inducible factors (HIFs). We demonstrate that HIFs mediate paracrine signaling between breast cancer cells (BCCs) and mesenchymal stem cells (MSCs) to promote metastasis. In a mouse orthotopic implantation model, MSCs were recruited to primary breast tumors and promoted BCC metastasis to LNs and lungs in a HIF-dependent manner. Coculture of MSCs with BCCs augmented HIF activity in BCCs. Additionally, coculture induced expression of the chemokine CXCL10 in MSCs and the cognate receptor CXCR3 in BCCs, which was augmented by hypoxia. CXCR3 expression was blocked in cocultures treated with neutralizing antibody against CXCL10. Conversely, CXCL10 expression was blocked in MSCs cocultured with BCCs that did not express CXCR3 or HIFs. MSC coculture did not enhance the metastasis of HIF-deficient BCCs. BCCs and MSCs expressed placental growth factor (PGF) and its cognate receptor VEGFR1, respectively, in a HIF-dependent manner, and CXCL10 expression by MSCs was dependent on PGF expression by BCCs. PGF promoted metastasis of BCCs and also facilitated homing of MSCs to tumors. Thus, HIFs mediate complex and bidirectional paracrine signaling between BCCs and MSCs that stimulates breast cancer metastasis.

Hypoxia-inducible factor (HIF) 1 and HIF-2 are heterodimeric proteins composed of an oxygen-regulated HIF-1α or HIF-2α subunit, respectively, and a constitutively expressed HIF-1β subunit, which mediate adaptive transcriptional responses to hypoxia. Here, we report that Sirt7 (sirtuin-7) negatively regulates HIF-1α and HIF-2α protein levels by a mechanism that is independent of prolyl hydroxylation and that does not involve proteasomal or lysosomal degradation. The effect of Sirt7 was maintained in the presence of the sirtuin inhibitor nicotinamide and upon deletion or mutation of its deacetylase domain, indicating a non-catalytic function. Knockdown of Sirt7 led to an increase in HIF-1α and HIF-2α protein levels and an increase in HIF-1 and HIF-2 transcriptional activity. Thus, we identify a novel molecular function of Sirt7 as a negative regulator of HIF signaling.

The advent of stem cell based therapies has brought regenerative medicine into an increased focus as a part of the modern medicine practice, with a potential to treat a myriad of intractable diseases in the future. Stem cells reside in a complex microenvironment presenting them with a multitude of potential cues that are chemical, physical, and mechanical in nature. Conventional techniques used for experiments involving stem cells can only poorly mimic the physiological context, and suffer from imprecise spatial and temporal control, low throughput, lack of scalability and reproducibility, and poor representation of the mechanical and physical cell microenvironment. Novel lab-on-a-chip platforms, on the other hand, can much better mimic the complexity of in vivo tissue milieu and provide a greater control of the parameter variation in a high throughput and scalable manner. This capability may be especially important for understanding the biology and cementing the clinical potential of stem cell based therapies. Here we review microfabrication- and microfluidics-based approaches to investigating the complex biology of stem cell responses to changes in the local microenvironment. In particular, we categorize each method based on the types of controlled inputs it can have on stem cells, including soluble biochemical factors, extracellular matrix interactions, homotypic and heterotypic cell-cell signaling, physical cues (e.g. oxygen tension, pH, temperature), and mechanical forces (e.g. shear, topography, rigidity). Finally, we outline the methods to perform large scale observations of stem cell phenotypes and high-throughput screening of cellular responses to a combination of stimuli, and many new emerging technologies that are becoming available specifically for stem cell applications.