Mimicking and analyzing the tumor microenvironment.

The tumor microenvironment (TME) is increasingly appreciated to play a decisive role in cancer development and response to therapy in all solid tumors. Hypoxia, acidosis, high interstitial pressure, nutrient-poor conditions, and high cellular heterogeneity of the TME arise from interactions between cancer cells and their environment. These properties, in turn, play key roles in the aggressiveness and therapy resistance of the disease, through complex reciprocal interactions between the cancer cell genotype and phenotype, and the physicochemical and cellular environment. Understanding this complexity requires the combination of sophisticated cancer models and high-resolution analysis tools. Models must allow both control and analysis of cellular and acellular TME properties, and analyses must be able to capture the complexity at high depth and spatial resolution. Here, we review the advantages and limitations of key models and methods in order to guide further TME research and outline future challenges.

NHE1 regulation in NAFLD in vitro contributes to hepatocyte injury and HSC crosstalk.

Non-alcoholic fatty liver disease (NAFLD) is the fastest-growing cause of liver-associated death globally. Whole-body knockout (KO) of Na+/H+ exchanger 1 (NHE1, SLC9A1) was previously proposed to protect against high-fat diet-induced liver damage; however, mechanistic insight was lacking. The aim of the present work was to address this question in vitro to determine how NHE1, specifically in hepatocytes, impacts lipid overload-induced inflammation, fibrosis, and hepatocyte-hepatic stellate cell (HSC) crosstalk. We induced palmitate (PA)-based steatosis in AML12 and HepG2 hepatocytes; manipulated NHE1 activity pharmacologically and by CRISPR/Cas9-mediated KO and overexpression; and measured intracellular pH (pHi), steatosis-associated inflammatory and fibrotic mediators, and cell death. PA treatment increased NHE1 mRNA levels but modestly reduced NHE1 protein expression and hepatocyte pHi. NHE1 KO in hepatocytes did not alter lipid droplet accumulation but reduced inflammatory signaling (p38 MAPK activity), lipotoxicity (4-HNE accumulation), and apoptosis (poly-ADP-ribose-polymerase-1 (PARP) cleavage). Conditioned medium from PA-treated hepatocytes increased the expression of NHE1 and of the fibrosis regulator tissue inhibitor of matrix metalloproteinases-2 in LX-2 HSCs, in a manner abolished by NHE1 KO in hepatocytes. We conclude that NHE1 is regulated in NAFLD in vitro and contributes to the ensuing damage by aggravating hepatocyte injury and stimulating hepatocyte-HSC crosstalk.

Adaptation to an acid microenvironment promotes pancreatic cancer organoid growth and drug resistance.

Harsh environments in poorly perfused tumor regions may select for traits driving cancer aggressiveness. Here, we investigated whether tumor acidosis interacts with driver mutations to exacerbate cancer hallmarks. We adapted mouse organoids from normal pancreatic duct (mN10) and early pancreatic cancer (mP4, KRAS-G12D mutation, ± p53 knockout) from extracellular pH 7.4 to 6.7, representing acidic niches. Viability was increased by acid adaptation, a pattern most apparent in wild-type (WT) p53 organoids, and exacerbated upon return to pH 7.4. This led to increased survival of acid-adapted organoids treated with gemcitabine and/or erlotinib, and, in WT p53 organoids, acid-induced attenuation of drug effects. New genetic variants became dominant during adaptation, yet they were unlikely to be its main drivers. Transcriptional changes induced by acid and drug adaptation differed overall, but acid adaptation increased the expression of gemcitabine resistance genes. Thus, adaptation to acidosis increases cancer cell viability after chemotherapy.

How protons pave the way to aggressive cancers.

Cancers undergo sequential changes to proton (H+) concentration and sensing that are consequences of the disease and facilitate its further progression. The impact of protonation state on protein activity can arise from alterations to amino acids or their titration. Indeed, many cancer-initiating mutations influence pH balance, regulation or sensing in a manner that enables growth and invasion outside normal constraints as part of oncogenic transformation. These cancer-supporting effects become more prominent when tumours develop an acidic microenvironment owing to metabolic reprogramming and disordered perfusion. The ensuing intracellular and extracellular pH disturbances affect multiple aspects of tumour biology, ranging from proliferation to immune surveillance, and can even facilitate further mutagenesis. As a selection pressure, extracellular acidosis accelerates disease progression by favouring acid-resistant cancer cells, which are typically associated with aggressive phenotypes. Although acid-base disturbances in tumours often occur alongside hypoxia and lactate accumulation, there is now ample evidence for a distinct role of H+-operated responses in key events underpinning cancer. The breadth of these actions presents therapeutic opportunities to change the trajectory of disease.

The prolactin receptor scaffolds Janus kinase 2 via co-structure formation with phosphoinositide-4,5-bisphosphate.

Class 1 cytokine receptors transmit signals through the membrane by a single transmembrane helix to an intrinsically disordered cytoplasmic domain that lacks kinase activity. While specific binding to phosphoinositides has been reported for the prolactin receptor (PRLR), the role of lipids in PRLR signaling is unclear. Using an integrative approach combining nuclear magnetic resonance spectroscopy, cellular signaling experiments, computational modeling, and simulation, we demonstrate co-structure formation of the disordered intracellular domain of the human PRLR, the membrane constituent phosphoinositide-4,5-bisphosphate (PI(4,5)P2) and the FERM-SH2 domain of the Janus kinase 2 (JAK2). We find that the complex leads to accumulation of PI(4,5)P2 at the transmembrane helix interface and that the mutation of residues identified to interact specifically with PI(4,5)P2 negatively affects PRLR-mediated activation of signal transducer and activator of transcription 5 (STAT5). Facilitated by co-structure formation, the membrane-proximal disordered region arranges into an extended structure. We suggest that the co-structure formed between PRLR, JAK2, and PI(4,5)P2 locks the juxtamembrane disordered domain of the PRLR in an extended structure, enabling signal relay from the extracellular to the intracellular domain upon ligand binding. We find that the co-structure exists in different states which we speculate could be relevant for turning signaling on and off. Similar co-structures may be relevant for other non-receptor tyrosine kinases and their receptors.

Dynamic localization of the Na+-HCO3- co-transporter NBCn1 to the plasma membrane, centrosomes, spindle and primary cilia.

Finely tuned regulation of transport protein localization is vital for epithelial function. The Na+-HCO3- co-transporter NBCn1 (also known as SLC4A7) is a key contributor to epithelial pH homeostasis, yet the regulation of its subcellular localization is not understood. Here, we show that a predicted N-terminal ß-sheet and short C-terminal α-helical motif are essential for NBCn1 plasma membrane localization in epithelial cells. This localization was abolished by cell-cell contact disruption, and co-immunoprecipitation (co-IP) and proximity ligation (PLA) revealed NBCn1 interaction with E-cadherin and DLG1, linking it to adherens junctions and the Scribble complex. NBCn1 also interacted with RhoA and localized to lamellipodia and filopodia in migrating cells. Finally, analysis of native and GFP-tagged NBCn1 localization, subcellular fractionation, co-IP with Arl13B and CEP164, and PLA of NBCn1 and tubulin in mitotic spindles led to the surprising conclusion that NBCn1 additionally localizes to centrosomes and primary cilia in non-dividing, polarized epithelial cells, and to the spindle, centrosomes and midbodies during mitosis. We propose that NBCn1 traffics between lateral junctions, the leading edge and cell division machinery in Rab11 endosomes, adding new insight to the role of NBCn1 in cell cycle progression.

Chronic acidosis rewires cancer cell metabolism through PPARα signaling.

The mechanisms linking tumor microenvironment acidosis to disease progression are not understood. Here, we used mammary, pancreatic, and colon cancer cells to show that adaptation to growth at an extracellular pH (pHe ) mimicking acidic tumor niches is associated with upregulated net acid extrusion capacity and elevated intracellular pH at physiological pHe , but not at acidic pHe . Using metabolic profiling, shotgun lipidomics, imaging and biochemical analyses, we show that the acid adaptation-induced phenotype is characterized by a shift toward oxidative metabolism, increased lipid droplet-, triacylglycerol-, peroxisome content and mitochondrial hyperfusion. Peroxisome proliferator-activated receptor-α (PPARA, PPARα) expression and activity are upregulated, at least in part by increased fatty acid uptake. PPARα upregulates genes driving increased mitochondrial and peroxisomal mass and ß-oxidation capacity, including mitochondrial lipid import proteins CPT1A, CPT2 and SLC25A20, electron transport chain components, peroxisomal proteins PEX11A and ACOX1, and thioredoxin-interacting protein (TXNIP), a negative regulator of glycolysis. This endows acid-adapted cancer cells with increased capacity for utilizing fatty acids for metabolic needs, while limiting glycolysis. As a consequence, the acid-adapted cells exhibit increased sensitivity to PPARα inhibition. We conclude that PPARα is a key upstream regulator of metabolic changes favoring cancer cell survival in acidic tumor niches.

Crosstalk between tumor acidosis, p53 and extracellular matrix regulates pancreatic cancer aggressiveness.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with minimal treatment options and a global rise in prevalence. PDAC is characterized by frequent driver mutations including KRAS and TP53 (p53), and a dense, acidic tumor microenvironment (TME). The relation between genotype and TME in PDAC development is unknown. Strikingly, when wild type (WT) Panc02 PDAC cells were adapted to growth in an acidic TME and returned to normal pH to mimic invasive cells escaping acidic regions, they displayed a strong increase of aggressive traits such as increased growth in 3-dimensional (3D) culture, adhesion-independent colony formation and invasive outgrowth. This pattern of acidosis-induced aggressiveness was observed in 3D spheroid culture as well as upon organotypic growth in matrigel, collagen-I and combination thereof, mimicking early and later stages of PDAC development. Acid-adaptation-induced gain of cancerous traits was further increased by p53 knockout (KO), but only in specific extracellular matrix (ECM) compositions. Akt- and Transforming growth factor-ß (TGFß) signaling, as well as expression of the Na+ /H+ exchanger NHE1, were increased by acid adaptation. Whereas Akt inhibition decreased spheroid growth regardless of treatment and genotype, stimulation with TGFßI increased growth of WT control spheroids, and inhibition of TGFß signaling tended to limit growth under acidic conditions only. Our results indicate that a complex crosstalk between tumor acidosis, ECM composition and genotype contributes to PDAC development. The findings may guide future strategies for acidosis-targeted therapies.

Acid Adaptation Promotes TRPC1 Plasma Membrane Localization Leading to Pancreatic Ductal Adenocarcinoma Cell Proliferation and Migration through Ca2+ Entry and Interaction with PI3K/CaM.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with a low overall survival rate of less than 10% and limited therapeutic options. Fluctuations in tumor microenvironment pH are a hallmark of PDAC development and progression. Many ion channels are bona fide cellular sensors of changes in pH. Yet, the interplay between the acidic tumor microenvironment and ion channel regulation in PDAC is poorly understood. In this study, we show that acid adaption increases PANC-1 cell migration but attenuates proliferation and spheroid growth, which are restored upon recovery. Moreover, acid adaptation and recovery conditions favor the plasma membrane localization of the pH-sensitive calcium (Ca2+) channel transient receptor potential C1 (TRPC1), TRPC1-mediated Ca2+ influx, channel interaction with the PI3K p85α subunit and calmodulin (CaM), and AKT and ERK1/2 activation. Knockdown (KD) of TRPC1 suppresses cell migration, proliferation, and spheroid growth, notably in acid-recovered cells. KD of TRPC1 causes the accumulation of cells in G0/G1 and G2/M phases, along with reduced expression of CDK6, -2, and -1, and cyclin A, and increased expression of p21CIP1. TRPC1 silencing decreases the basal Ca2+ influx in acid-adapted and -recovered cells, but not in normal pH conditions, and Ca2+ chelation reduces cell migration and proliferation solely in acid adaptation and recovery conditions. In conclusion, acid adaptation and recovery reinforce the involvement of TRPC1 in migration, proliferation, and cell cycle progression by permitting Ca2+ entry and forming a complex with the PI3K p85α subunit and CaM.

The TRPC1 Channel Forms a PI3K/CaM Complex and Regulates Pancreatic Ductal Adenocarcinoma Cell Proliferation in a Ca2+-Independent Manner.

Dysregulation of the transient receptor canonical ion channel (TRPC1) has been found in several cancer types, yet the underlying molecular mechanisms through which TRPC1 impacts pancreatic ductal adenocarcinoma (PDAC) cell proliferation are incompletely understood. Here, we found that TRPC1 is upregulated in human PDAC tissue compared to adjacent pancreatic tissue and this higher expression correlates with low overall survival. TRPC1 is, as well, upregulated in the aggressive PDAC cell line PANC-1, compared to a duct-like cell line, and its knockdown (KD) reduced cell proliferation along with PANC-1 3D spheroid growth by arresting cells in the G1/S phase whilst decreasing cyclin A, CDK2, CDK6, and increasing p21CIP1 expression. In addition, the KD of TRPC1 neither affected Ca2+ influx nor store-operated Ca2+ entry (SOCE) and reduced cell proliferation independently of extracellular calcium. Interestingly, TRPC1 interacted with the PI3K-p85α subunit and calmodulin (CaM); both the CaM protein level and AKT phosphorylation were reduced upon TRPC1 KD. In conclusion, our results show that TRPC1 regulates PDAC cell proliferation and cell cycle progression by interacting with PI3K-p85α and CaM through a Ca2+-independent pathway.

Metabolic reprogramming by driver mutation-tumor microenvironment interplay in pancreatic cancer: new therapeutic targets.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers globally with a mortality rate exceeding 95% and very limited therapeutic options. A hallmark of PDAC is its acidic tumor microenvironment, further characterized by excessive fibrosis and depletion of oxygen and nutrients due to poor vascularity. The combination of PDAC driver mutations and adaptation to this hostile environment drives extensive metabolic reprogramming of the cancer cells toward non-canonical metabolic pathways and increases reliance on scavenging mechanisms such as autophagy and macropinocytosis. In addition, the cancer cells benefit from metabolic crosstalk with nonmalignant cells within the tumor microenvironment, including pancreatic stellate cells, fibroblasts, and endothelial and immune cells. Increasing evidence shows that this metabolic rewiring is closely related to chemo- and radioresistance and immunosuppression, causing extensive treatment failure. Indeed, stratification of human PDAC tumors into subtypes based on their metabolic profiles was shown to predict disease outcome. Accordingly, an increasing number of clinical trials target pro-tumorigenic metabolic pathways, either as stand-alone treatment or in conjunction with chemotherapy. In this review, we highlight key findings and potential future directions of pancreatic cancer metabolism research, specifically focusing on novel therapeutic opportunities.

The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells.

The SCN4B gene, coding for the NaVß4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVß4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVß4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of ß-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navß4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.

Dynamic Na+/H+ exchanger 1 (NHE1) - calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation.

Calmodulin (CaM) engages in Ca2+-dependent interactions with numerous proteins, including a still incompletely understood physical and functional interaction with the human Na+/H+-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts stably expressing wildtype and mutant NHE1, we discovered multiple accessible states of this functionally important complex existing in different NHE1:CaM stoichiometries and structures. We determined the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities could be tuned by variations in NHE1:CaM ratio and calcium ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding α-helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation and by mutation of the first CaM-binding α-helix, whereas it was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results demonstrate a diversity of NHE1:CaM interaction modes and suggest that CaM may contribute to NHE1 dimerization and thereby augment NHE1 regulation. We propose that a similar structural diversity is of relevance to many other CaM complexes.

Putting Warburg to work: how imaging of tumour acidosis could help predict metastatic potential in breast cancer.

Solid tumours are often highly acidic compared to normal tissue, and tumour extracellular acidosis contributes to multiple aspects of cancer progression. Now, Anemone et al. in this issue of the British Journal of Cancer provide in vivo evidence that the degree to which various breast cancer cell lines acidify their environment correlates with their ability to metastasise to the lungs. This indicates that measurements of tumour extracellular acidosis have the potential to become a clinical tool for assessing the risk of metastasis.

Cancer Cell Acid Adaptation Gene Expression Response Is Correlated to Tumor-Specific Tissue Expression Profiles and Patient Survival.

The acidic pH of the tumor microenvironment plays a critical role in driving cancer development toward a more aggressive phenotype, but the underlying mechanisms are unclear. To this end, phenotypic and genotypic changes induced by adaptation of cancer cells to chronic acidosis have been studied. However, the generality of acid adaptation patterns across cell models and their correlation to the molecular phenotypes and aggressiveness of human cancers are essentially unknown. Here, we define an acid adaptation expression response shared across three cancer cell models, dominated by metabolic rewiring, extracellular matrix remodeling, and altered cell cycle regulation and DNA damage response. We find that many genes which are upregulated by acid adaptation are significantly correlated to patient survival, and more generally, that there are clear correlations between acid adaptation expression response and gene expression change between normal and tumor tissues, for a large subset of cancer patients. Our data support the notion that tumor microenvironment acidity is one of the key factors driving the selection of aggressive cancer cells in human patient tumors, yet it also induces a growth-limiting genotype that likely limits cancer cell growth until the cells are released from acidosis, for instance during invasion.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells.

Increased metabolic acid production and upregulation of net acid extrusion render pH homeostasis profoundly dysregulated in many cancers. Plasma membrane activity of vacuolar H+ ATPases (V-ATPases) has been implicated in acid extrusion and invasiveness of some cancers, yet often on the basis of unspecific inhibitors. Serving as a membrane anchor directing V-ATPase localization, the a subunit of the V0 domain of the V-ATPase (ATP6V0a1-4) is particularly interesting in this regard. Here, we map the regulation and roles of ATP6V0a3 in migration, invasion, and growth in pancreatic ductal adenocarcinoma (PDAC) cells. a3 mRNA and protein levels were upregulated in PDAC cell lines compared to non-cancer pancreatic epithelial cells. Under control conditions, a3 localization was mainly endo-/lysosomal, and its knockdown had no detectable effect on pHi regulation after acid loading. V-ATPase inhibition, but not a3 knockdown, increased HIF-1 expression and decreased proliferation and autophagic flux under both starved and non-starved conditions, and spheroid growth of PDAC cells was also unaffected by a3 knockdown. Strikingly, a3 knockdown increased migration and transwell invasion of Panc-1 and BxPC-3 PDAC cells, and increased gelatin degradation in BxPC-3 cells yet decreased it in Panc-1 cells. We conclude that in these PDAC cells, a3 is upregulated and negatively regulates migration and invasion, likely in part via effects on extracellular matrix degradation.

The γ-hydroxybutyric acid (GHB) analogue NCS-382 is a substrate for both monocarboxylate transporters subtypes 1 and 4.

The small-molecule ligand (E)-2-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is an analogue of γ-hydroxybutyric acid (GHB) and is widely used for probing the brain-specific GHB high-affinity binding sites. To reach these, brain uptake is imperative, and it is therefore important to understand the molecular mechanisms of NCS-382 transport in order to direct in vivo studies. In this study, we hypothesized that NCS-382 is a substrate for the monocarboxylate transporter subtype 1 (MCT1) which is known to mediate blood-brain barrier (BBB) permeation of GHB. For this purpose, we investigated NCS-382 uptake by MCT subtypes endogenously expressed in tsA201 and MDA-MB-231 cell lines in assays of radioligand-based competition and fluorescence-based intracellular pH measurements. To further verify the results, we measured NCS-382 uptake by means of mass spectrometry in Xenopus laevis oocytes heterologously expressing MCT subtypes. As expected, we found that NCS-382 is a substrate for MCT1 with half-maximal effective concentrations in the low millimolar range. Surprisingly, NCS-382 also showed substrate activity at MCT4 as well as uptake in water-injected oocytes, suggesting a component of passive diffusion. In conclusion, transport of NCS-382 across membranes differs from GHB as it also involves MCT4 and/or passive diffusion. This should be taken into consideration when designing pharmacological studies with this compound and its closely related analogues. The combination of MCT assays used here exemplifies a setup that may be suitable for a reliable characterization of MCT ligands in general.

Annual Meeting of the International Society of Cancer Metabolism (ISCaM): Metabolic Adaptations and Targets in Cancer.

The metabolism of cancer cells differs from that of their normal counterparts in a spectrum of attributes, including imbalances in diverse metabolic arms and pathways, metabolic plasticity and extent of adaptive responses, levels, and activities of metabolic enzymes and their upstream regulators and abnormal fluxes of metabolic intermediates and products. These attributes endow cancer cells with the ability to survive stressors of the tumor microenvironment and enable them to landscape and exploit the host terrain, thereby facilitating cancer progression and therapy resistance. Understanding the molecular and physiological principles of cancer metabolism is one of the key prerequisites for the development of better anticancer treatments. Therefore, various aspects of cancer metabolism were addressed at the 5th annual meeting of the International Society of Cancer Metabolism (ISCaM) in Bratislava, Slovakia, on October 17-20, 2018. The meeting presentations and discussions were traditionally focused on mechanistic, translational, and clinical characteristics of metabolism and pH control in cancer, at the level of molecular pathways, cells, tissues, and organisms. In order to reflect major healthcare challenges of the current era, ISCaM has extended its scope to metabolic disorders contributing to cancer, as well as to opportunities for their prevention, intervention, and therapeutic targeting.

Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker proteins in brain endothelial cells/astrocyte co-cultures.

Ischemic stroke has been shown to induce breakdown of the blood-brain barrier, although these changes are not fully characterized. Oxygen-glucose deprivation (OGD) has been used to investigate the effects of ischemia in cultured brain capillary endothelial cells, however this involves a change of medium which in itself may affect the cells. The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes. Barrier properties were evaluated by transendothelial electrical resistance measurements, passive permeability of flux markers, RT-qPCR and immunocytochemistry. Both OGD and simple medium exchange caused an increase in endothelial monolayer permeability. This correlated with reduced transcript levels of a number of tight junction and tight junction-associated proteins (claudin-1, claudin-5, occludin, ZO-1, tricellulin, marveld3 and PECAM-1), as well as with altered transcript level of several transporters and receptors (GLUT-1, HB-EGF, InsR, TfR, two members of the low density lipoprotein receptor family, LDLR and LRP-1, and the efflux transporter BCRP). In contrast, effects induced specifically by OGD were transient de-localization of claudin-5 from the junction zone, increased InsR localization at the plasma membrane and transient downregulation of MRP-1 and P-gp transcript levels. In conclusion, OGD caused changes in claudin-5 and InsR localization, as well as in MRP-1 and P-gp transcript levels. Our results however also indicated that medium exchange alone caused changes in functional barrier properties and expression levels of wide range of proteins.

HER2 and p95HER2 differentially regulate miRNA expression in MCF-7 breast cancer cells and downregulate MYB proteins through miR-221/222 and miR-503.

The HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR. miRNAs strongly regulated by p95HER2 yet not by HER2, included miR-221, miR-222, miR-503, miR-29a, miR-149, miR-196 and miR-361. Estrogen receptor-α (ESR1) expression was essentially ablated by p95HER2 expression, in a manner recapitulated by miR-221/-222 mimics. c-Myb family transcription factors MYB and MYBL1, but not MYBL2, were downregulated by p95HER2 and by miR-503 or miR-221/-222 mimics. MYBL1 3'UTR inhibition by miR-221/222 was lost by deletion of a single putative miR-221/222 binding sites. p95HER2 expression, or knockdown of either MYB protein, elicited upregulation of tissue inhibitor of matrix metalloprotease-2 (TIMP2). miR-221/222 and -503 mimics increased, and TIMP2 knockdown decreased, cell migration and invasion. A similar pathway was operational in T47D- and SKBr-3 cells. This work reveals important differences between HER2- and p95HER2- mediated miRNA changes in breast cancer cells, provides novel mechanistic insight into regulation of MYB family transcription factors by p95HER2, and points to a role for a miR-221/222- MYB family-TIMP2 axis in regulation of motility in breast cancer cells.