Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction.

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium.

Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is a leading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

MARCH2, a Novel Oncogene-regulated SNAIL E3 Ligase, Suppresses Triple-negative Breast Cancer Metastases.

Epithelial-mesenchymal transition (EMT) in cancer promotes metastasis and chemotherapy resistance. A subset of triple-negative breast cancer (TNBC) exhibits a mesenchymal gene signature that is associated with poor patient outcomes. We previously identified PTK6 tyrosine kinase as an oncogenic driver of EMT in a subset of TNBC. PTK6 induces EMT by stabilizing SNAIL, a key EMT-initiating transcriptional factor. Inhibition of PTK6 activity reverses mesenchymal features of TNBC cells and suppresses their metastases by promoting SNAIL degradation via a novel mechanism. In the current study, we identify membrane-associated RING-CH2 (MARCH2) as a novel PTK6-regulated E3 ligase that promotes the ubiquitination and degradation of SNAIL protein. The MARCH2 RING domain is critical for SNAIL ubiquitination and subsequent degradation. PTK6 inhibition promotes the interaction of MARCH2 with SNAIL. Overexpression of MARCH2 exhibits tumor suppressive properties and phenocopies the effects of SNAIL downregulation and PTK6 inhibition in TNBC cells, such as inhibition of migration, anoikis resistance, and metastasis. Consistent with this, higher levels of MARCH2 expression in breast and other cancers are associated with better prognosis. We have identified MARCH2 as a novel SNAIL E3 ligase that regulates EMT and metastases of mesenchymal TNBC. SIGNIFICANCE: EMT is a process directly linked to drug resistance and metastasis of cancer cells. We identified MARCH2 as a novel regulator of SNAIL, a key EMT driver, that promotes SNAIL ubiquitination and degradation in TNBC cells. MARCH2 is oncogene regulated and inhibits growth and metastasis of TNBC. These insights could contribute to novel strategies to therapeutically target TNBC.

Dendritic cell-mediated responses to secreted Cryptosporidium effectors promote parasite-specific CD8+ T cell responses.

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells, type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium.

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction.

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. The use of single cell RNA sequencing to profile IEC during infection revealed induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells, and IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ demonstrated the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ-mediated bystander activation of uninfected enterocytes is important for control of Cryptosporidium.

Dendritic cell-mediated responses to secreted Cryptosporidium effectors are required for parasite-specific CD8 + T cell responses.

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the MHC-I restricted SIINFEKL epitope which is recognized by TCR transgenic OT-I CD8 + T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8 + T cells that were a source of IFN-γ that could restrict growth of Cryptosporidium . This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (ROP1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells (IEC), type I dendritic cells (cDC1) were required to generate CD8 + T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as targets of the immune system and suggest that crosstalk between enterocytes and cDC1s is crucial for CD8 + T cell responses to Cryptosporidium .

Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on fecal shedding of new oocysts. The entire life cycle thus occurs in a single host and features asexual as well as sexual forms of replication. Here, we identify and locus tag two Apetala 2-type (AP2) transcription factors and demonstrate that they are exclusively expressed in male and female gametes, respectively. To enable functional studies of essential genes in Cryptosporidium parvum, we develop and validate a small-molecule-inducible gene excision system, which we apply to the female factor AP2-F to achieve conditional gene knockout. Analyzing this mutant, we find the factor to be dispensable for asexual growth and early female fate determination in vitro but to be required for oocyst shedding in infected animals in vivo. Transcriptional analyses conducted in the presence or absence of AP2-F revealed that the factor controls the transcription of genes encoding crystalloid body proteins, which are exclusively expressed in female gametes. In C. parvum, the organelle is restricted to sporozoites, and its loss in other apicomplexan parasites leads to blocked transmission. Overall, our development of conditional gene ablation in C. parvum provides a robust method for genetic analysis in this parasite that enabled us to identify AP2-F as an essential regulator of transcription required for oocyst shedding and transmission. IMPORTANCE The parasite Cryptosporidium infects millions of people worldwide each year, leading to life-threatening diarrheal disease in young children and immunosuppressed individuals. There is no vaccine and only limited treatment. Transmission occurs via the fecal-oral route by an environmentally resilient spore-like oocyst. Infection takes place in the intestinal epithelium, where parasites initially propagate asexually before transitioning to male and female gametes, with sex leading to the formation of new oocysts. The essential role of sexual development for continuous infection and transmission makes it an attractive target for therapy and prevention. To study essential genes and potential drug targets across the life cycle, we established inducible gene excision for C. parvum. We determined that the female-specific transcription factor AP2-F is not required for asexual growth and early female development in vitro but is necessary for oocyst shedding in vivo. This work enhances the genetic tools available to study Cryptosporidium gene function.

PRKCQ inhibition enhances chemosensitivity of triple-negative breast cancer by regulating Bim.

BACKGROUND: Protein kinase C theta, (PRKCQ/PKCθ) is a serine/threonine kinase that is highly expressed in a subset of triple-negative breast cancers (TNBC) and promotes their growth, anoikis resistance, epithelial-mesenchymal transition (EMT), and invasion. Here, we show that PRKCQ regulates the sensitivity of TNBC cells to apoptosis triggered by standard-of-care chemotherapy by regulating levels of pro-apoptotic Bim. METHODS: To determine the effects of PRKCQ expression on chemotherapy-induced apoptosis, shRNA and cDNA vectors were used to modulate the PRKCQ expression in MCF-10A breast epithelial cells or triple-negative breast cancer cells (MDA-MB231Luc, HCC1806). A novel PRKCQ small-molecule inhibitor, 17k, was used to inhibit kinase activity. Viability and apoptosis of cells treated with PRKCQ cDNA/shRNA/inhibitor +/-chemotherapy were measured. Expression levels of Bcl2 family members were assessed. RESULTS: Enhanced expression of PRKCQ is sufficient to suppress apoptosis triggered by paclitaxel or doxorubicin treatment. Downregulation of PRKCQ also enhanced the apoptosis of chemotherapy-treated TNBC cells. Regulation of chemotherapy sensitivity by PRKCQ mechanistically occurs via regulation of levels of Bim, a pro-apoptotic Bcl2 family member; suppression of Bim prevents the enhanced apoptosis observed with combined PRKCQ downregulation and chemotherapy treatment. Regulation of Bim and chemotherapy sensitivity is significantly dependent on PRKCQ kinase activity; overexpression of a catalytically inactive PRKCQ does not suppress Bim or chemotherapy-associated apoptosis. Furthermore, PRKCQ kinase inhibitor treatment suppressed growth, increased anoikis and Bim expression, and enhanced apoptosis of chemotherapy-treated TNBC cells, phenocopying the effects of PRKCQ downregulation. CONCLUSIONS: These studies support PRKCQ inhibition as an attractive therapeutic strategy and complement to chemotherapy to inhibit the growth and survival of TNBC cells.

PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells.

BACKGROUND: The protein kinase C (PKC) family comprises distinct classes of proteins, many of which are implicated in diverse cellular functions. Protein tyrosine kinase C theta isoform (PRKCQ)/PKCθ, a member of the novel PKC family, may have a distinct isoform-specific role in breast cancer. PKCθ is preferentially expressed in triple-negative breast cancer (TNBC) compared to other breast tumor subtypes. We hypothesized that PRKCQ/PKCθ critically regulates growth and survival of a subset of TNBC cells. METHODS: To elucidate the role of PRKCQ/PKCθ in regulating growth and anoikis resistance, we used both gain and loss of function to modulate expression of PRKCQ. We enhanced expression of PKCθ (kinase-active or inactive) in non-transformed breast epithelial cells (MCF-10A) and assessed effects on epidermal growth factor (EGF)-independent growth, anoikis, and migration. We downregulated expression of PKCθ in TNBC cells, and determined effects on in vitro and in vivo growth and survival. TNBC cells were also treated with a small molecule inhibitor to assess requirement for PKCθ kinase activity in the growth of TNBC cells. RESULTS: PRKCQ/PKCθ can promote oncogenic phenotypes when expressed in non-transformed MCF-10A mammary epithelial cells; PRKCQ/PKCθ enhances anchorage-independent survival, growth-factor-independent proliferation, and migration. PKCθ expression promotes retinoblastoma (Rb) phosphorylation and cell-cycle progression under growth factor-deprived conditions that typically induce cell-cycle arrest of MCF-10A breast epithelial cells. Proliferation and Rb phosphorylation are dependent on PKCθ-stimulated extracellular signal-related kinase (Erk)/mitogen-activated protein kinase (MAPK) activity. Enhanced Erk/MAPK activity is dependent on the kinase activity of PKCθ, as overexpression of kinase-inactive PKCθ does not stimulate Erk/MAPK or Rb phosphorylation or promote growth-factor-independent proliferation. Downregulation of PRKCQ/PKCθ in TNBC cells enhances anoikis, inhibits growth in 3-D MatrigelTM cultures, and impairs triple-negative tumor xenograft growth. AEB071, an inhibitor of PKCθ kinase activity, also inhibits growth and invasive branching of TNBC cells in 3-D cultures, further supporting a role for PKCθ kinase activity in triple-negative cancer cell growth. CONCLUSIONS: Enhanced PRKCQ/PKCθ expression can promote growth-factor-independent growth, anoikis resistance, and migration. PRKCQ critically regulates growth and survival of a subset of TNBC. Inhibition of PKCθ kinase activity may be an attractive therapeutic approach for TNBC, a subtype in need of improved targeted therapies.

PTK6 Inhibition Suppresses Metastases of Triple-Negative Breast Cancer via SNAIL-Dependent E-Cadherin Regulation.

Patients with triple-negative breast cancers (TNBC) are at high risk for recurrent or metastatic disease despite standard treatment, underscoring the need for novel therapeutic targets and strategies. Here we report that protein tyrosine kinase 6 (PTK6) is expressed in approximately 70% of TNBCs where it acts to promote survival and metastatic lung colonization. PTK6 downregulation in mesenchymal TNBC cells suppressed migration and three-dimensional culture growth, and enhanced anoikis, resistance to which is considered a prerequisite for metastasis. PTK6 downregulation restored E-cadherin levels via proteasome-dependent degradation of the E-cadherin repressor SNAIL. Beyond being functionally required in TNBC cells, kinase-active PTK6 also suppressed E-cadherin expression, promoted cell migration, and increased levels of mesenchymal markers in nontransformed MCF10A breast epithelial cells, consistent with a role in promoting an epithelial-mesenchymal transition (EMT). SNAIL downregulation and E-cadherin upregulation mediated by PTK6 inhibition induced anoikis, leading to impaired metastatic lung colonization in vivo Finally, effects of PTK6 downregulation were phenocopied by treatment with a recently developed PTK6 kinase inhibitor, further implicating kinase activity in regulation of EMT and metastases. Our findings illustrate the clinical potential for PTK6 inhibition to improve treatment of patients with high-risk TNBC. Cancer Res; 76(15); 4406-17. ©2016 AACR.