Garcia-Areas, Ramon A.

It is estimated that one in eight women will be diagnosed with breast cancer. Developing an
understanding of the tumor microenvironment is critical for developing treatments for breast cancer
patients. It has been well established that hypoxia, or a lack of oxygen, is fundamental in creating a
microenvironment that enables metastasis via eliciting angiogenic processes. Poorly differentiated blood
vessels can fashion an oxygen-deprived microenvironment that triggers the expression of transcription
factor Hypoxia Inducible Factor alpha HIF-1alpha that in turn can up regulate genes mediating a protumor
effect. Our laboratory has discovered that mammary tumors express Semaphorin7a SEMA7A, a
HIF-1alpha inducible protein. This study’s objective is to delineate the mechanism for hypoxia induction
in mammary cells. Cobalt Chloride CoCl2 was used to mimic hypoxia in mammary tumor cell cultures.
Flow cytometry was used to determine HIF-1alpha activity in the mammary cells. Benign EpH4 mammary
cells expressing low SEMA7A greatly increased its expression after response to hypoxic stimuli as
determined by qPCR and SEMA7A-specific ELISA. Pretreatment of EpH4 cells with a HIF-1alpha
inhibitor Chemotin blocked induction of SEMA7A. Paradoxically, CoCl2 did not raise expression in the
highly metastatic 4T1 cells which experience high levels of SEMA7A. Treatment of 4T1 cells with
Chemotin under normoxic conditions inhibit HIF-1alpha activity and decrease SEMA7A levels.
Determining the role of SEMA7A in the hypoxia axis could further elucidate novel pathways in breast
cancer, suggesting that malignant tumor cells can utilize HIF-1alpha in a hypoxic independent manner.

Solid tumors can hijack many of the same programs used in neurogenesis
to enhance tumor growth and metastasis, thereby generating a plethora of
neurogenesis-related molecules including semaphorins Among them, we have
identified Semaphorin7A (SEMA7A) in breast cancer We first used to the DA-3
mammary tumor model to determine the effect of tumor-derived SEMA7A on
immune cells We found that tumor-derived SEMA7A can modulate the
production of proangiogenic chemokines CXCL2/MIP-2 and CXCL 1, and prometastatic
MMP-9 in macrophages We next aimed to determine the expression
and function of SEMA7A in mammary tumor cells We found that SEMA7A is
highly expressed in both metastatic human and murine breast cancer cells We
show that both TGF-β and hypoxia elicits the production of SEMA 7 A in mammary
cells SEMA7 A shRNA silencing in 4T1 cells resulted in decreased mesenchymal
markers MMP-3, MMP-13, Vimentin and TGF-β) SEMA7A silenced cells show increased stiffness with reduced migratory and proliferative potential In vivo,
SEMA7A silenced 4T1 tumor bearing mice showed decreased tumor growth and
metastasis Genetic ablation of host-derived SEMA7A synergized to further
decrease the growth and metastasis of 4T1 cells Our findings suggest novel
functional roles for SEMA7A in breast cancer and that SEMA7A could be a novel
therapeutic target to limit tumor growth and metastasis